Visualizing Viral Onslaught: How Cryo-Electron Tomography Reveals Host-Pathogen Battles in 3D

Addison Parker Jan 12, 2026 474

This article provides a comprehensive guide to Cryo-Electron Tomography (cryo-ET) for imaging host-virus interactions at molecular resolution.

Visualizing Viral Onslaught: How Cryo-Electron Tomography Reveals Host-Pathogen Battles in 3D

Abstract

This article provides a comprehensive guide to Cryo-Electron Tomography (cryo-ET) for imaging host-virus interactions at molecular resolution. It explores the fundamental principles of cryo-ET, detailing its methodological pipeline from sample vitrification to sub-tomogram averaging. We address key challenges in specimen preparation and data acquisition, offering troubleshooting strategies for optimal results. The article critically validates cryo-ET against other structural biology techniques, highlighting its unique ability to capture viruses and cellular machinery in their native, functional states. Aimed at virologists, structural biologists, and drug discovery professionals, this resource underscores cryo-ET's transformative role in identifying novel antiviral targets and advancing therapeutic development.

Beyond Snapshots: Unpacking the Core Principles of Cryo-ET for Native-State Virology

Application Note: Imaging Host-Virus Interactions in Situ

Cryo-Electron Tomography (cryo-ET) enables the visualization of macromolecular complexes within their native cellular environment at sub-nanometer resolution. This application note details its use for studying the life cycle of viruses—from cellular entry and replication to assembly and egress—directly within infected host cells, providing mechanistic insights unobtainable by other methods.

Key Quantitative Advantages of Cryo-ET for Host-Virus Research

Table 1: Resolution and Throughput Comparison of Structural Techniques

Technique Typical Resolution for Cellular Targets Sample Preparation Environment Key Advantage for Virology
Cryo-ET 2-4 Å (targets), 20-40 Å (in situ) Vitrification, thinning (FIB/SEM) Native, hydrated cellular context Visualizes virus structure inside the cell
Single-Particle Cryo-EM 1.5-3.5 Å Purified virus particles Isolated, buffer conditions Atomic models of symmetric capsids
X-ray Crystallography 1.0-3.0 Å Crystallized proteins/virions Crystal lattice Highest resolution for ordered complexes
Confocal Light Microscopy ~250 nm Fluorescent tagging Live or fixed cells Dynamics and tracking over time

Table 2: Recent Cryo-ET Studies of Notable Viruses (2022-2024)

Virus Family Host Cell Type Key Biological Insight (Resolved In Situ) Approx. Resolution Reference Type
Herpesviridae Human epithelial Tegument protein organization during capsid assembly & egress 26 Å Nature 2023
Coronaviridae (SARS-CoV-2) Vero E6 Spike conformation on virion, viral RNA packing, double-membrane vesicles 31 Å Cell 2022
HIV-1 Human lymphocytes Capsid lattice structure within the nucleus 4.3 Å (capsid) Science 2023
Influenza A MDCK Membrane fusion protein dynamics 28 Å PNAS 2024

Protocol: Cryo-ET Workflow for Imaging Virus-Infected Cells

Part 1: Cell Culture, Infection, and Cryo-Preparation

Objective: To preserve virus-infected cells in a native, frozen-hydrated state for tomographic imaging.

Materials & Reagents:

  • Virus of interest (e.g., SARS-CoV-2, HSV-1)
  • Permissive host cell line (e.g., Huh-7, Vero, HEK293)
  • Transmission Electron Microscopy (TEM) grids (200-300 mesh gold Quantifoil R2/2 or R2/1)
  • Plasma cleaner (e.g., Gatan Solarus)
  • Cryo-plunger (e.g., Vitrobot Mark IV)
  • Liquid ethane/propane mixture
  • Growth media & virus dilution media

Procedure:

  • Grid Preparation: Plasma clean gold TEM grids for 20-30 seconds to render them hydrophilic.
  • Cell Culture and Infection: Seed cells onto grids placed in a multi-well plate. Allow to adhere (4-6 hrs). Infect grids at desired MOI for a specific time point post-infection. Include uninfected control grids.
  • Vitrification: Using the Vitrobot, blot excess media from the grid for 2-4 seconds at 100% humidity and plunge-freeze into liquid ethane cooled by liquid nitrogen. Store grids under liquid nitrogen.

Part 2: Focused Ion Beam (FIB) Milling for Lamella Preparation

Objective: To create an electron-transparent lamella (~100-200 nm thick) of the infected cell.

Materials & Reagents:

  • Cryo-FIB/SEM microscope (e.g., Thermo Fisher Scientific Scios 2HYDRA or TFS Aquilos 2)
  • Cryo-transfer station and shuttle
  • Organometallic platinum or carbon gas injection system (GIS)

Procedure:

  • Transfer: Load the vitrified grid into the cryo-FIB/SEM under liquid nitrogen conditions.
  • Sputter Coating: Apply a uniform organometallic platinum layer (~1-2 µm) via GIS to protect the cell surface.
  • Rough Milling: Using a high ion beam current (∼1 nA), mill trenches on either side of the target region (e.g., a cell showing cytopathic effect).
  • Fine Milling & Polishing: Reduce the current sequentially (to 100 pA, then 50 pA) to thin the lamella to the target thickness. Final polish at low current (10 pA) to reduce amorphous damage.
  • Verification: Image the lamella using the SEM beam at 2-5 kV to assess quality and thickness.

Part 3: Cryo-ET Data Acquisition and Reconstruction

Objective: To collect a tilt series and reconstruct a 3D tomogram of the lamella.

Materials & Reagents:

  • 300 kV Cryo-TEM equipped with a direct electron detector (e.g., Thermo Fisher Krios G4 with Falcon 4 or Gatan K3)
  • Software: SerialEM, Tomo5, or EPU for automated data collection.
  • Processing Software: IMOD, AreTomo, EMAN2, Warp, M.

Procedure:

  • Grid Mapping: Insert the lamella into the cryo-TEM. Acquire low-magnification maps to locate promising lamellae.
  • Tilt Series Acquisition: Using SerialEM, target a region of interest. Collect a single-axis tilt series from -60° to +60° with a 2° or 3° increment, under dose-fractionated mode. Use a total dose of 80-150 e⁻/Ų. Defocus range: -3 to -8 µm.
  • Motion Correction & Alignment: Use MotionCor2 or the detector's integrated software for frame alignment. Align tilt images using fiducial-less (patch-tracking) methods in IMOD or AreTomo.
  • Reconstruction: Generate a 3D reconstruction via weighted back-projection or SIRT-like algorithms in IMOD or M.
  • Denoising & Segmentation: Apply deep learning-based denoising (e.g., IsoNet, cryoCARE). Manually or semi-automatically segment features of interest (virions, organelles, filaments) using Amira or EMAN2.

Visualizations

G a Virus Infection of Host Cells on TEM Grid b Rapid Plunge-Freezing (Vitrification) a->b c Targeted Milling (Cryo-FIB/SEM) b->c d Tilt-Series Acquisition in Cryo-TEM c->d e 3D Tomogram Reconstruction d->e f Subtomogram Averaging & Classification e->f g Atomic Modeling & Interpretation f->g

Cryo-ET Workflow for Host-Virus Imaging

G virion Virion Attachment & Membrane Fusion endosome Endosomal Trafficking virion->endosome receptor Host Cell Surface Receptor receptor->virion capsid Capsid Uncoating & Genome Release endosome->capsid genome Viral Replication & Transcription capsid->genome assembly Virion Assembly & Maturation genome->assembly egress Egress via Exocytosis/Lysis assembly->egress

Viral Life Cycle Stages Visualized by Cryo-ET

The Scientist's Toolkit: Essential Reagents & Materials

Table 3: Key Research Reagent Solutions for Cryo-ET Virology

Item Function in Cryo-ET Workflow Example Product/Note
Gold TEM Grids (Holey Carbon) Support film for cell growth and imaging. Gold is inert and conducts charge. Quantifoil Au R2/2, 200 mesh
Cryogen (Liquid Ethane) Rapidly vitrifies aqueous samples to preserve native state without ice crystals. >99.9% purity, mixed with propane for stability.
Organometallic Pt Gas (GIS) Deposits a protective layer on the cell surface prior to FIB milling, preventing damage. Trimethyl(methylcyclopentadienyl)platinum(IV)
Fiducial Gold Beads (Optional) Provide reference points for aligning tilt series images. BSA-treated 10nm colloidal gold.
Cryo-TEM Autoloader Maintains grid at <-170°C during transfer into the microscope column. Thermo Fisher Autogrid.
Direct Electron Detector Captures dose-fractionated images with high detective quantum efficiency (DQE). Gatan K3, Falcon 4.
Cellular Cryo-Fluid (Culture Media) Maintains cell viability and correct osmotic balance during blotting and freezing. Often serum-free to reduce background.
Anti-contamination Cold Trap Prevents condensation of contaminants onto the sample during TEM imaging. Integral part of the TEM column.

Within the broader thesis on Cryo-Electron Tomography (Cryo-ET) for imaging host-virus interactions, understanding the fundamental workflow from 2D projections to a 3D tomogram is paramount. This process enables the visualization of viral entry, replication, and egress in a near-native, frozen-hydrated state at molecular resolution. This Application Note details the protocols and principles underlying tomographic reconstruction, a core technique for structural cell biology and antiviral drug discovery.

The Tomographic Imaging Workflow: A Step-by-Step Protocol

The generation of a 3D tomogram from 2D projections involves a sequential pipeline of specimen preparation, data acquisition, and computational reconstruction.

Protocol 2.1: Cryo-Electron Tomography Sample Preparation and Data Acquisition

Objective: To acquire a tilt series of 2D projections from a vitrified cellular sample containing host-virus complexes.

Materials & Reagents:

  • Cultured host cells (e.g., HEK293, Vero E6) infected with virus of interest.
  • Quantifoil or C-flat holey carbon EM grids.
  • Vitrification device (e.g., Thermo Fisher Scientific Vitrobot Mark IV).
  • 300 keV Cryo-Transmission Electron Microscope equipped with a cryo-holder, energy filter (Gatan GIF), and direct electron detector (e.g., Gatan K3, Falcon 4).
  • Acquisition software (e.g., SerialEM, Tomography 5.0).

Procedure:

  • Specimen Vitrification: Apply 3-4 µL of the infected cell suspension, or lamella from a focused ion beam (FIB)-milled cellular volume, to a glow-discharged EM grid. Blot excess liquid and plunge-freeze the grid into liquid ethane using the Vitrobot to achieve vitreous ice.
  • Microscope Setup: Insert the cryo-holder into the TEM. Align the microscope for parallel illumination and set the desired dose (e.g., 80-120 e⁻/Ų total for the series).
  • Tilt Series Acquisition: a. Locate a region of interest at low magnification. b. Using acquisition software, define a tilt scheme (typically from -60° to +60° with 1-3° increments). c. Initiate automated acquisition. The software will tilt the specimen, track the area, and focus (using dose-symmetric or predictive methods), recording one 2D projection image at each tilt angle. d. The total electron dose is fractionated across all tilt images to minimize radiation damage.

Protocol 2.2: Tomographic Reconstruction and 3D Volume Generation

Objective: To computationally align the 2D tilt series and reconstruct a 3D tomogram.

Materials & Software:

  • Tilt series stack (e.g., .mrc, .tiff format).
  • Processing software: IMOD, EMAN2, Scipion, or Warp.
  • High-performance GPU-accelerated workstation.

Procedure:

  • Pre-processing: Import the tilt series. Apply motion correction and dose-weighting if not done on-the-fly. Generate a contrast transfer function (CTF) model for each image.
  • Alignment: a. Fiducial-less (Patch-Tracking) Alignment: The software divides images into patches, tracks their movement across tilts, and computes a geometric model to align all projections to a common origin. b. Fiducial-Based Alignment: If gold beads are present, their 3D coordinates are used to compute precise alignment parameters.
  • Reconstruction: a. Use the alignment parameters to back-project the 2D information into 3D space. b. Apply a Weighted Back-Projection (WBP) or Simultaneous Iterative Reconstruction Technique (SIRT) algorithm to compute the initial 3D volume (tomogram).
  • Post-processing: Apply denoising algorithms (e.g., IsoNet, Cryo-CARE) to enhance the signal-to-noise ratio for interpretation and segmentation.

Data Presentation: Key Parameters in Cryo-ET Workflow

Table 1: Quantitative Parameters for Cryo-ET Tilt Series Acquisition

Parameter Typical Range Impact on Final Tomogram
Accelerating Voltage 200 - 300 keV Higher voltage increases penetration, crucial for thicker cellular samples.
Total Electron Dose 80 - 120 e⁻/Ų Limits radiation damage; must be fractionated across all tilts.
Tilt Range ±50° to ±70° Larger range reduces the "missing wedge" of information.
Tilt Increment 1° - 3° Finer increments improve reconstruction fidelity at cost of dose/time.
Pixel Size 2 - 5 Å/pixel Defines the sampling resolution; finer pixels increase data size.
Defocus -3 to -8 µm Chosen to balance contrast and interpretable resolution.

Table 2: Comparison of Reconstruction Algorithms

Algorithm Principle Advantages Limitations
Weighted Back Projection (WBP) Direct back-projection of 2D images with weighting to compensate for missing wedge. Fast, deterministic, good for initial assessment. Amplifies noise, artifacts from missing wedge more pronounced.
Simultaneous Iterative Reconstruction Technique (SIRT) Iterative method minimizing discrepancy between calculated and actual projections. Superior noise suppression, yields cleaner volumes. Computationally intensive, iterative parameters require tuning.

Visualizing the Workflow

G cluster_prep 1. Specimen Preparation cluster_acq 2. Data Acquisition cluster_rec 3. Reconstruction cluster_ana 4. Analysis Cell Infected Cells Vit Vitrification (Plunge-Freezing) Cell->Vit Grid Cryo-EM Grid Vit->Grid TEM Cryo-TEM Tilt Series Grid->TEM Proj Set of 2D Projection Images TEM->Proj Align Alignment (Patch Tracking) Proj->Align Recon 3D Reconstruction (WBP/SIRT) Align->Recon Tomo 3D Tomogram Recon->Tomo Denoise Denoising & Segmentation Tomo->Denoise Model Molecular Interpretation Denoise->Model

Diagram Title: Cryo-ET Workflow from Specimen to 3D Model

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Research Reagent Solutions for Host-Virus Cryo-ET

Item Function in Cryo-ET Workflow
Holey Carbon EM Grids (Quantifoil, C-flat) Support film with holes to suspend vitrified cellular material across a vacuum, enabling imaging.
Focused Ion Beam (FIB) / Scanning Electron Microscope (SEM) Used to mill thin (200-300 nm) lamellae from vitrified cells, providing access to internal structures for tomography.
Direct Electron Detector (e.g., Gatan K3, Falcon 4) Captures 2D projection images with high detective quantum efficiency (DQE) and fast frame rates, enabling dose-fractionation.
Gold Fiducial Beads (10-15 nm) Often added to sample to serve as reference markers for precise alignment of tilt series images.
Cryo-TEM Holder Maintains specimen at cryogenic temperatures (< -170°C) in the microscope column to prevent ice crystallization and reduce radiation damage.
Denoising Software (IsoNet, Cryo-CARE) AI/ML-based tools that significantly enhance the signal-to-noise ratio in tomograms, revealing macromolecular details.
Subtomogram Averaging Software (RELION, emClarity) Aligns and averages thousands of copies of a particle extracted from a tomogram to achieve high-resolution 3D structures.

Within the broader thesis on Cryo-Electron Tomography (Cryo-ET) for host-virus interaction research, vitrification is the foundational, enabling technique. It allows the rapid freezing of biological samples in their native, hydrated state, trapping transient molecular events—like viral entry, replication complex formation, and progeny assembly—in a thin layer of non-crystalline, "vitreous" ice. This process preserves high-fidelity structural information for subsequent Cryo-ET imaging.

Table 1: Comparison of Key Vitrification Methods for Cryo-ET Sample Preparation

Method Plunge Freezing High-Pressure Freezing (HPF) Jet/Vitreous Sectioning
Sample Thickness Limit < 5 µm (optimal < 0.3 µm) Up to 200 µm Can be applied to HPF samples, sectioned to 100-300 nm
Cooling Rate ~10^5 K/s (at surface) ~10^4 K/s (under high pressure) N/A (freezing done prior)
Ice Crystal Artifact Risk Low for thin edges, high for thick regions Very low throughout volume Risk of knife marks & compression
Primary Application in Host-Virus Research Purified viruses, viral particles on cell surfaces, thin cellular projections. Infected cell monolayers or small tissue chunks. Thick tissues or cell pellets from infection models.
Typical Throughput High (grids per minute) Low (minutes per sample) Low (hours per ribbon)
Key Advantage for Transient Events Ultra-rapid freezing of surface events. Excellent preservation of internal cellular architecture during infection. Enables tomography of infected tissue architecture.

Detailed Protocol: Plunge Freezing for Viral Entry Studies on Cell Periphery

This protocol details vitrification of virus-bound cells for capturing early entry steps.

I. Materials & Pre-Vitrification

  • Cultured cells (e.g., HAP1, HeLa) grown on plasma-cleaned, gold Quantifoil or UltrauFoil EM grids.
  • Virus preparation: Purified virus particles at high titer (>10^8 pfu/mL) in appropriate, non-volatile buffer.
  • Vitrification device: e.g., Thermo Fisher Scientific Vitrobot Mark IV or Leica EM GP.
  • Liquid ethane: Generated by condensing ethane gas in a small cup cooled by liquid nitrogen.
  • Blotting paper: Standard Vitrobot filter paper (Grade 595).
  • Forceps and storage boxes: Pre-cooled, autoclaved tweezers and grid storage boxes.

II. Infection & Vitrification

  • Incubation: Transfer the grid with confluent cells to a 37°C, 5% CO₂ humidified chamber. Apply 3-5 µL of virus inoculum directly onto the cell-coated side. Incubate for the precise time point desired (e.g., 2-5 minutes for early attachment/entry).
  • Blotting & Plunging:
    • Quickly retrieve the grid and, using the vitrification device, blot from the back (grid side) for 1-3 seconds with specified blot force (typically 0-5) to remove excess liquid, leaving a thin film.
    • Immediately plunge the grid at maximum speed into liquid ethane cooled by liquid nitrogen. Total time from blot to freezing should be <250 ms.
  • Storage: Transfer the vitrified grid under liquid nitrogen to a pre-labeled grid box for storage until Cryo-ET data collection.

Detailed Protocol: High-Pressure Freezing & Freeze Substitution for Assembly Site Analysis

For thicker samples like infected cells, HPF followed by freeze-substitution (FS) and plastic embedding can precede tomography (Cryo-CET) or be used for correlative light and electron microscopy (CLEM).

I. Materials

  • HPF machine: e.g., Leica EM ICE or Wohlwend Compact 03.
  • Sample Carriers: Type A (200 µm deep) or B (100 µm deep) carriers.
  • Hexadecene or Yeast paste as a filler/inert cryoprotectant.
  • FS Medium: 1% Osmium Tetroxide, 0.1% Uranyl Acetate in anhydrous acetone.
  • FS apparatus: Automated freeze-substitution unit (e.g., Leica AFS2).

II. Protocol

  • Loading: Grow cells on a suitable substrate (e.g., sapphire discs). Infect at desired MOI. At the target time point, quickly sandwich the disc with cells facing a flat carrier, filled with hexadecene. Load into HPF machine.
  • High-Pressure Freezing: Trigger freezing. Sample experiences 2100 bar pressure and is frozen by jetting liquid nitrogen at ~10^4 K/s.
  • Freeze-Substitution (FS): Under liquid nitrogen, transfer frozen sample to a pre-cooled (-90°C) vial containing FS medium.
    • Run FS program: -90°C for 72 hours, warm to -20°C at 5°C/hour, hold at -20°C for 2 hours, warm to 4°C at 10°C/hour, then to 20°C.
  • Embedding & Sectioning: Wash in acetone, infiltrate with epoxy resin (e.g., Epon), polymerize. Section (70-300 nm) for tomography.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Vitrification in Host-Virus Cryo-ET

Item Function & Rationale
UltrauFoil Holey Gold Grids (R1.2/1.3) Gold grids provide superior thermal conductivity for faster freezing. UltrauFoil's pre-defined hole pattern increases yield of usable, thin ice areas over cells.
Plasma Cleaner (e.g., Gatan Solarus) Renders grids hydrophilic, ensuring even sample spread and thin ice formation, critical for imaging virus-cell contact sites.
Fiducial Gold Beads (10-15 nm) Essential for tomogram alignment during data processing. Added to sample just before blotting/plunging.
CryoProtectants (e.g., 10% Dextran) For plunge freezing, can be mixed with virus inoculum to improve ice quality by reducing water content, though may affect biological activity.
HPF Carriers with Cavities Enable freezing of infected cell monolayers or small tissue explants with minimal crushing, preserving 3D spatial relationships of viral factories.
Correlative Fluorescent Dyes (e.g., CellMask, GFP) For CLEM. Allows targeting of specific infected cells or cellular compartments for Cryo-ET, linking dynamic fluorescence to high-resolution structure.

workflow cluster_vir Viral Event Capture Strategy V1 Synchronized Infection (Precise MOI & Timing) V2 Rapid Quenching (Vitrification) V1->V2 S2 Apply Virus (Incubate for target time) V1->S2 S4 Plunge into Liquid Ethane V2->S4 S1 Sample Preparation (Cells on EM Grids) S1->S2 S3 Blot Excess Liquid S2->S3 S3->S4 S5 Grid Storage in LN₂ S4->S5 S6 Cryo-ET Data Collection S5->S6

Vitrification Workflow for Viral Events

hierarchy Thesis Thesis: Cryo-ET of Host-Virus Interactions Vit Core Technique: Vitrification (Trapping Native State) Thesis->Vit Sample Sample Type & Biological Question Vit->Sample SP1 Thin (<5µm) (e.g., Virus, Cell Periphery) Sample->SP1 SP2 Thick (>5µm) (e.g., Infected Cells, Tissue) Sample->SP2 Method Vitrification Method SP1->Method SP2->Method M1 Plunge Freezing Method->M1 M2 High-Pressure Freezing Method->M2 Output Output for Cryo-ET M1->Output M2->Output O1 Thin Vitrified Layer on Grid Output->O1 O2 Vitrified Bulk Sample (For sectioning or Cryo-CET) Output->O2

Method Selection Based on Sample

Application Notes

Cryo-Electron Tomography (cryo-ET) enables the high-resolution 3D visualization of macromolecular complexes within their native cellular environment. This technique is pivotal for host-virus interaction research, as it allows for the direct observation of viral entry, replication, assembly, and egress processes without the artifacts induced by chemical fixation, dehydration, or staining. The key advantage is the preservation of cellular ultrastructure in a near-native, vitrified state, enabling the imaging of macromolecules in situ at sub-nanometer resolution.

Within the thesis context of studying host-virus interactions, cryo-ET provides an unparalleled spatial and structural context. Researchers can directly visualize viral glycoproteins engaging host cell receptors, the formation of viral replication organelles, and the assembly of virions in crowded cellular compartments. This direct visualization is crucial for understanding mechanistic details and identifying vulnerable points for therapeutic intervention, directly informing rational drug and vaccine design.

Recent advancements (2023-2024) have been accelerated by integrated workflows combining focused ion beam (FIB) milling and cryo-ET. The development of plasma FIB (pFIB) systems, notably using xenon, has significantly increased the throughput and quality of lamella preparation from vitrified cells. Furthermore, the integration of advanced phase plates and direct electron detectors, coupled with new software for template matching and subtomogram averaging, now allows for the precise localization and structural determination of complexes even within dense cellular matrices.

Table 1: Comparative Analysis of In Situ Structural Techniques

Technique Approximate Resolution Sample Preparation Key Advantage for Host-Virus Studies Primary Limitation
Cryo-ET (with FIB milling) 2-4 Å (STA), ~20-40 Å (tomogram) Vitrification, FIB milling Visualizes complexes in native cellular context. Sample thickness limitation; requires lamella preparation.
Single-Particle Cryo-EM 1.5-3.5 Å Purification, Vitrification Achieves highest resolution for purified complexes. Removes all cellular context.
Cryo-Correlative Light & EM (CLEM) ~20-50 nm (FM), ~20-40 Å (ET) Vitrification, Fluorescent tagging Targets rare or specific events for ET. Resolution of fluorescence channel is low.
Serial Block-Face SEM 5-50 nm Chemical Fixation, Dehydration, Staining Large volume 3D ultrastructure. Non-native state; no molecular details.

Table 2: Key Performance Metrics in Modern Cryo-ET Workflows (2023-2024)

Workflow Step Technology/Method Typical Duration Success Rate/Output
Vitrification Automated plunge freezer (e.g., Vitrobot, CP3) 1-2 hours (prep + freezing) >95% ice quality consistency
Lamella Preparation Xenon Plasma FIB (e.g., Thermo Scientific Tomē) 2-4 hours per lamella 5-8 high-quality lamellae per session
Data Acquisition 300 keV FEG with DED & Phase Plate (e.g., K3/GIF) 8-12 hours per tomogram series 40-60 tomograms per 24-hour session
Data Processing Template Matching & Subtomogram Averaging (Warp, M, RELION) 1-5 days (depending on volume) Can resolve complexes to 3-5 Å in situ

Experimental Protocols

Protocol 1: Cryo-FIB Milling for Lamella Preparation from Virus-Infected Cells

Objective: To produce an electron-transparent lamella (~200 nm thick) from a vitrified virus-infected cell for cryo-ET imaging.

Materials: See "The Scientist's Toolkit" below.

Procedure:

  • Cell Culture & Infection: Grow host cells (e.g., HEK293, Vero) on glow-discharged gold EM grids. Infect with virus at a high MOI (e.g., 5-10) for a predetermined optimal time.
  • Vitrification: Using a plunge freezer, blot the grid to a thin layer of liquid and rapidly plunge into liquid ethane. Store in liquid nitrogen.
  • Cryo-CLEM (Optional): Transfer grid to a cryo-fluorescence microscope. Identify and map cells expressing a fluorescently tagged viral protein or showing infection morphology.
  • FIB Milling Setup: Load the grid into a dual-beam FIB/SEM microscope equipped with a cryo-stage. Sputter-coat the sample with an organometallic platinum or iridium gas to enhance conductivity and protect the surface.
  • Rough Milling: Using the ion beam (30 kV, 1-3 nA), mill large trenches on either side of the target region (identified by CLEM or SEM imaging).
  • Fine Milling & Polishing: Gradually reduce the ion beam current (to 50-100 pA) to thin the lamella to the target thickness of 150-250 nm. Use the electron beam for endpoint monitoring to avoid over-milling.
  • Lamella Transfer: Using a cryo-scanning manipulator, weld a micromanipulator needle to the lamella, cut it free, and transfer it to a specialized cryo-TEM grid (AutoGrid or similar).
  • Storage: Store the prepared lamella grid under liquid nitrogen until data collection.

Protocol 2: Cryo-ET Data Acquisition and Reconstruction

Objective: To collect a tilt series of a cellular lamella and reconstruct a 3D tomogram.

Procedure:

  • Microscope Setup: Load the lamella grid into a 200-300 keV cryo-TEM equipped with a direct electron detector (DED) and energy filter (slit width 20 eV).
  • Screening & Targeting: At low magnification (~1,000x), locate the lamella. At higher mag (~5,000x), identify a region of interest (ROI) devoid of milling artifacts.
  • Tilt Series Acquisition: Using automated software (e.g., SerialEM, Tomography 5):
    • Align the microscope eucentric height at the ROI.
    • Define a tilt scheme (typically from -60° to +60° with 2-3° increments).
    • Set a cumulative electron dose limit of 80-120 e⁻/Ų, distributed across all tilts.
    • Initiate acquisition. The software will collect a movie at each tilt angle, with beam-induced motion correction via the DED.
  • Tomogram Reconstruction:
    • Motion Correction & Alignment: Use software like MotionCor2 or Warp to align frames within each tilt movie.
    • Tilt Series Alignment: Align the tilt images using fiducial markers (gold beads) or patch-tracking algorithms (e.g., in IMOD).
    • Reconstruction: Compute a 3D reconstruction using weighted back-projection or SIRT-like algorithms (in IMOD or Astra) to generate the tomogram.

Diagrams

G Cell Virus-Infected Cell on EM Grid Vit High-Pressure Plunge Freezing Cell->Vit CryoCell Vitrified Cell (Hydrated, Native State) Vit->CryoCell FIB Cryo-FIB Milling (Lamella Preparation) CryoCell->FIB Lamella Electron-Transparent Lamella (~200 nm) FIB->Lamella ET Cryo-ET Acquisition (Tilt Series) Lamella->ET Tomo 3D Tomogram (~20-40 Å Resolution) ET->Tomo Proc Subtomogram Averaging & Analysis Tomo->Proc Structure In Situ Atomic Model of Host-Virus Complex Proc->Structure

Workflow for In Situ Cryo-ET Structure Determination

H cluster_host Host Cell cluster_virus Virus Particle H1 Surface Receptor H2 Endosomal Membrane V2 Viral Capsid H2->V2 2. Uncoating H3 Cytosolic Factor H4 Viral Replication Organelle (RO) V1 Viral Glycoprotein (Prefusion State) V1->H1 1. Attachment & Fusion V3 Viral Genome V3->H4 3. Genome Replication V4 Viral Polymerase Complex V4->H3 4. Assembly & Egress

Key Host-Virus Interaction Stages Visualized by Cryo-ET

The Scientist's Toolkit

Table 3: Essential Research Reagents & Materials for Cryo-ET of Host-Virus Interactions

Item Function & Importance Example Product/Type
Quantifoil or C-flat Grids Gold or copper grids with a perforated carbon film. Provide support for cells while leaving large areas suspended for imaging. Quantifoil R2/2, 200 mesh Au.
Plunge Freezer Instrument for ultra-rapid cooling of samples to form vitreous (non-crystalline) ice, preserving native structure. Thermo Fisher Vitrobot Mark IV, Leica GP2.
Cryo-FIB/SEM Microscope Dual-beam microscope for milling thin lamellae from vitrified cells under cryo-conditions. Essential for in situ work. Thermo Scientific Tomē, Teneo VolumeScope.
Cryo-Transmission EM High-voltage TEM with cryo-stage and direct electron detector for high-resolution, low-dose imaging of tilt series. Thermo Scientific Krios G4, Glacios 2.
Direct Electron Detector (DED) Camera capable of counting individual electrons. Crucial for low-dose imaging and motion correction. Gatan K3, Falcon 4.
Volta Phase Plate Increases contrast in cryo-ET images, allowing lower electron doses and better visualization of fine cellular details. Thermo Scientific Set of 4.
Subtomogram Averaging Software Software packages for aligning and averaging thousands of extracted subvolumes to achieve high-resolution in situ structures. RELION, M, Warp.
Cellular EM Media Specialized buffers for plunge freezing that maintain physiological conditions and promote thin ice. DMEM with HEPES, PBS with FBS.
Cryogenic Storage Dewars For safe, long-term storage of vitrified grids and lamellae in liquid nitrogen. Taylor-Wharton, custom racks.

Application Notes

The evolution of transmission electron microscopy (TEM) specimen preparation, culminating in high-pressure freezing (HPF), represents a pivotal advancement for structural biology, particularly in cryo-electron tomography (cryo-ET) of host-virus interactions. Early TEM methods relied on chemical fixation, dehydration, and staining, which introduced artifacts and failed to preserve native cellular architecture. The development of plunge-freezing in the 1980s enabled the vitrification of thin samples, but was inadequate for thicker, bulk biological specimens like eukaryotic cells infected with viruses. High-pressure freezing, commercialized in the 1990s, overcomes this by applying ~2100 bar pressure while rapidly cooling samples, suppressing ice crystal formation and allowing vitrification of samples up to ~200 µm thick. This is critical for cryo-ET, which requires a near-native, vitrified state to visualize macromolecular complexes, viral entry mechanisms, and replication factories in situ at molecular resolution.

Key Quantitative Evolution of TEM Specimen Preparation:

Table 1: Evolution of Key Parameters in TEM Specimen Preparation Techniques

Technique (Era) Primary Fixation Max. Vitrification Depth Temporal Resolution Key Artifact Introduced
Chemical Fixation (1940s-) Aldehydes (e.g., Glutaraldehyde) N/A Minutes to Hours Membrane extraction, protein aggregation, shrinkage.
Plunge Freezing (1980s-) Physical (Vitrification) ~1 µm (aqueous layer) Milliseconds (surface) Ice crystals in samples >1µm, preferential orientation.
High-Pressure Freezing (1990s-) Physical (Vitrification under HP) ~200 µm ~20-50 ms Compressional damage (rare), sample size restrictions.

Table 2: Impact on Host-Virus Interaction Research Parameters

Preparation Method Preservation of Macromolecular Complexes Suitability for Cellular Tomography Compatibility with CLEM* Throughput (Sample Prep)
Chemical Fixation/Plastic Embedding Low-Moderate (denatured) Low (severe artifacts) Moderate (post-processing) High
Plunge Freezing (Cryo-EM Grids) High Low (only thin edges/virions) Challenging Moderate
High-Pressure Freezing & Freeze-Substitution Moderate-High High (for plastic sections) High Low-Moderate
HPF & Cryo-FIB-milling for Cryo-ET Highest (near-native) Highest (in-situ vitrified cells) High (correlative workflow) Low

*CLEM: Correlative Light and Electron Microscopy.

Experimental Protocols

Protocol 1: High-Pressure Freezing of Virus-Infected Cell Monolayers for Cryo-FIB-milling and Cryo-ET

Objective: To vitrify a cultured cell monolayer infected with virus for subsequent preparation of a thin lamella via cryo-Focused Ion Beam (FIB) milling and analysis by cryo-ET.

Materials & Reagent Solutions: The Scientist's Toolkit: Key Research Reagent Solutions

Item Function in Protocol
EM-grade Hexadecene An inert, non-volatile cryoprotectant that fills intercellular spaces to improve heat conduction during HPF.
Type A or B Gold HPF Carriers 3mm diameter metal carriers with a 100-200µm cavity to hold the sample. Type B often has a flat and a well side.
Yeast Paste Used as a filler/backing material in the HPF carrier to provide uniform pressure and prevent sample collapse.
Liquid Nitrogen Primary cryogen for cooling and long-term storage of vitrified samples at -196°C.
High-Pressure Freezer (e.g., Leica EM ICE, Wohlwend HPF Compact 02) Apparatus to apply high pressure and rapidly jet-cool the sample.
Cryo-vials and Storage Dewars For secure, organized storage of frozen carriers under liquid nitrogen.

Methodology:

  • Cell Culture & Infection: Grow appropriate host cells (e.g., mammalian epithelial cells) on a plasma-cleaned, fibronectin-coated Type B gold HPF carrier (flat side). Infect cells with virus at desired MOI and for a specific time window post-infection.
  • Loading: At the time point of interest, gently rinse cells in carrier with room-temperature culture medium. Quickly blot the carrier edge and fill the cavity (cells facing inward) with a layer of hexadecene. Assemble the "sandwich" by placing a second, empty carrier (well-side facing the cells) on top.
  • High-Pressure Freezing: Immediately load the carrier sandwich into the HPF machine. Trigger freezing. The process applies ~2100 bar pressure and propels the sample into liquid nitrogen-cooled ethane or propane at cooling rates >20,000°C/sec.
  • Storage & Retrieval: Under liquid nitrogen, disassemble the sandwich using pre-cooled tools. Retrieve the carrier containing the frozen cell monolayer. Store it in a cryo-vial within a liquid nitrogen Dewar.
  • Downstream Processing: For cryo-ET, the frozen carrier is transferred to a cryo-FIB/SEM microscope. The frozen cell block is then sputter-coated and a ~150-300 nm thin lamella is milled across virus-infected cells using a Ga+ ion beam. This lamella is then imaged by cryo-ET.

Protocol 2: Freeze-Substitution and Embedding of HPF Samples for Conventional TEM

Objective: To prepare a chemically fixed, plastic-embedded sample from an HPF-vitrified specimen for ultrastructural analysis or immuno-EM.

Methodology:

  • Freeze-Substitution: Under LN2, transfer the vitrified HPF sample to a freeze-substitution apparatus (e.g., Leica EM AFS2) containing a solution of 1-2% osmium tetroxide, 0.1-0.5% uranyl acetate, and 5% water in anhydrous acetone. Maintain at -90°C for 48-72 hours.
  • Temperature Ramp: Gradually raise the temperature (e.g., 5°C/hour) to -20°C, hold for several hours, then raise to 4°C.
  • Washing & Infiltration: Rinse samples several times with anhydrous acetone at 4°C. Infiltrate with increasing concentrations of epoxy resin (e.g., Epon/Araldite) in acetone over 24-48 hours.
  • Polymerization: Place samples in fresh resin in molds and polymerize at 60°C for 48 hours.
  • Sectioning & Imaging: Ultramicrotome sections (70-90 nm) are collected, stained with lead citrate, and imaged in a TEM at 80-120 kV.

Diagrams

G EarlyTEM Early TEM (Chemical Fixation) PlungeFreeze Plunge Freezing (c. 1980s) EarlyTEM->PlungeFreeze Need for Native State HPF High-Pressure Freezing (c. 1990s) PlungeFreeze->HPF Need for Thick Samples CryoFIB Cryo-FIB Milling (c. 2000s) HPF->CryoFIB Need for Thin Lamellae CryoET Modern Cryo-ET Workflow CryoFIB->CryoET Enables In-Situ Tomography

Title: Evolution of TEM Specimen Preparation for Cryo-ET

G Culture Culture & Infect Cells on HPF Carrier HPFStep High-Pressure Freezing (Vitrification) Culture->HPFStep Storage LN2 Storage HPFStep->Storage PathA Path A: Cryo-FIB-milling Storage->PathA PathB Path B: Freeze-Substitution Storage->PathB CryoFIB Sputter Coat & Mill Lamella PathA->CryoFIB FS Osmium/UA in Acetone (-90°C to 4°C) PathB->FS CryoTomo Cryo-Tomography Data Collection CryoFIB->CryoTomo Embed Resin Infiltration & Polymerization FS->Embed TEM Ultramicrotomy & TEM Imaging Embed->TEM

Title: HPF Protocol Workflow for Host-Virus Imaging

A Step-by-Step Pipeline: Applying Cryo-ET to Image Viral Entry, Replication, and Egress

Cryo-electron tomography (cryo-ET) has emerged as a premier technique for visualizing the intricate structural details of host-virus interactions in a near-native, frozen-hydrated state. The core challenge, however, lies in preparing specimens thin enough (typically <300 nm) for electrons to traverse while preserving the complex cellular architecture and the dynamic, nanoscale events of viral entry, replication, and egress. Focused Ion Beam (FIB) milling at cryogenic temperatures is the established method for producing these thin lamellae from vitrified, infected cells. This application note details current, optimized protocols for generating high-quality lamellae from virus-infected cells, a critical step in the pipeline for structural cell biology and antiviral drug discovery.

Key Quantitative Parameters for High-Quality Lamella Generation

The success of FIB-milling is governed by several interdependent parameters. The following tables summarize optimal and critical ranges based on recent literature and technical advancements.

Table 1: Critical FIB-Milling Parameters for Infected Cell Lamellae

Parameter Optimal Range Purpose & Rationale
Accelerating Voltage 30 kV (for bulk milling), 5-8 kV (for final polishing) Higher voltage for faster sputtering; lower voltage reduces Ga+ ion implantation and amorphous damage layer.
Beam Current 1 nA (rough milling), 50-100 pA (fine milling/polishing) Higher currents for rapid material removal; lower currents for precise, clean finishes.
Lamella Target Thickness 150 - 250 nm Balance between electron transparency (cryo-ET) and structural integrity of the cellular volume.
Final Polish Milling Pattern Serpentine or meander, with <5 nm step size Produces a uniform surface, minimizing curtaining artifacts.
Working Distance 5 - 8 mm Optimizes resolution and depth of field for the electron beam imaging.
Tilt Angle (Pre-tilt) 8° - 12° (relative to ion beam) Corrects for the inherent wedge shape created by milling, aiming for parallel-sided lamella.

Table 2: Cell Culture & Vitrification Parameters for Infection Studies

Parameter Recommendation Rationale
Cell Confluence on EM Grid 70-90% Ensures isolated cells for milling while providing ample cell-cell contact zones often relevant for viral spread.
Multiplicity of Infection (MOI) 2 - 10 (time-point dependent) Optimizes for observing a sufficient number of infection events without excessive cellular disruption.
Post-Infection Time Point Dictated by viral lifecycle Synchronization is key (e.g., early times for entry, mid-times for replication, late for egress).
Vitrification Method High-pressure freezing or plunge-freezing (with blotting optimization) HPF preserves thicker samples (e.g., cell monolayers); plunge-freezing is standard for grid-grown cells.
Cryo-Protectant (for plunge-freezing) None, or low-concentration glycerol/sucrose (culture medium-based) Maintains native state; small additives can sometimes improve vitrification for thicker regions.

Detailed Experimental Protocol: FIB-Milling of Vitrified Infected Cells

Protocol 3.1: Pre-Milling Preparation of Vitrified Grids

  • Cell Culture & Infection: Grow appropriate host cells (e.g., HEK293, Huh-7, primary macrophages) directly on glow-discharged, gold EM grids (e.g., R2/2, R2/1, or holey carbon) placed in a multi-well plate. Infect cells at the desired MOI in a minimal infection volume. At the target time point, proceed immediately to vitrification.
  • Vitrification by Plunge-Freezing: Using a vitrification device (e.g., Vitrobot, EM GP):
    • Blot the grid from the back side for 3-5 seconds at 100% humidity, 37°C (for mammalian cells), then plunge into liquid ethane slush.
    • Critical: Optimize blotting time/force to achieve a thin, vitrified ice layer while retaining cell integrity.
  • Grid Storage & Transfer: Store grids under liquid nitrogen. Use a cryo-transfer shuttle to load the grid into the FIB-SEM microscope without warming.

Protocol 3.2: Cryo-FIB Lift-Out and Milling (Standard Workflow)

This protocol assumes a dual-beam FIB-SEM microscope (e.g., Thermo Scientific Aquilos 2, Teneo, or similar) equipped with a cryo-stage and a micromanipulator.

  • Microscope Setup: Insert the cryo-shuttle. Condense the cryo-stage anti-contaminator. Stabilize the grid temperature to below -170°C.
  • Localization: Use the SEM at low beam energy (2-5 kV) to navigate to a region of interest (ROI)—an infected cell displaying moderate cytopathic effect or a cell-cell junction.
  • Deposit Protective Layer: Use the Gas Injection System (GIS) to deposit a ~1-2 µm thick layer of organometallic platinum or carbon precursor on the ROI surface using the electron beam (E-beam) first, followed by ion-beam (I-beam) cross-linking for a robust coat.
  • Rough Milling: With the ion beam at 30 kV and 1 nA, mill two large, deep trenches on either side of the protected ROI. Rotate the stage to bring the lamella perpendicular to the ion beam (typically a 7-10° pre-tilt relative to the stage). Mill the backside of the lamella to a thickness of ~2-3 µm.
  • Lift-Out: Weld a cryo-manipulator needle to the top of the lamella using ion-beam deposited platinum. Cut the lamella free from its base. Carefully translate and weld the lamella onto a dedicated cryo-TEM grid (e.g., a half-moon grid or an Autogrid).
  • Thinning (Final Milling): Detach the needle. With the lamella on the new grid, perform sequential milling at reduced ion currents (300 pA -> 100 pA -> 50 pA). At the final step (50 pA or lower), use a polishing pattern to achieve the target thickness of 150-250 nm. Continuously monitor thickness using the SEM or by measuring the ion beam milling time at a known sputter rate.
  • Final Check & Storage: Capture a low-dose SEM overview image. Retract the stage and transfer the grid under cryogenic conditions to a storage box or directly to a cryo-TEM for tomographic data collection.

Diagrams: Workflows and Relationships

G CellInfection Cell Culture & Infection on EM Grid Vitrification Rapid Vitrification (Plunge-Freezing) CellInfection->Vitrification CryoTransfer Cryo-Transfer to FIB-SEM Vitrification->CryoTransfer ROI_Identification SEM Imaging & ROI Identification CryoTransfer->ROI_Identification PtDeposition E-/I-beam Deposition of Protective Layer ROI_Identification->PtDeposition RoughMilling Rough Milling (30 kV, 1 nA) PtDeposition->RoughMilling LiftOut Lamella Lift-Out & Transfer to TEM Grid RoughMilling->LiftOut FineMilling Sequential Fine Milling (100 pA -> 50 pA) LiftOut->FineMilling CryoET Cryo-TEM Imaging & Tomography FineMilling->CryoET

Diagram Title: Cryo-FIB Lamella Preparation Workflow for Infected Cells

G cluster_KeyChallenge Key Challenge: Resolving Host-Virus Dynamics cluster_Solution Solution: Cryo-ET on FIB-Milled Lamellae cluster_Outcome Research Outcome Challenge Need for High-Resolution 3D Imaging in Native State Sol1 Preserves Native Cellular Context Challenge->Sol1 Addresses via Sol2 Targets Specific Infection Events Challenge->Sol2 Addresses via Sol3 Enables Molecular-Scale Imaging of Interactions Challenge->Sol3 Addresses via Out1 Visualize Viral Entry Mechanisms Sol1->Out1 Out2 Map Replication Organelle Structure Sol2->Out2 Out3 Characterize Virion Assembly & Egress Sol3->Out3

Diagram Title: Rationale for Cryo-FIB in Host-Virus Research

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagents and Materials for Cryo-FIB of Infected Cells

Item Function & Specific Recommendation
Gold EM Grids (e.g., Quantifoil R2/2, Au 200 mesh) Substrate for cell growth. Gold is inert and conducts charge, preventing charging during FIB/SEM imaging.
Plunge Freezing Device (e.g., Vitrobot Mark IV, EM GP) Instrument for rapid vitrification of grid-grown infected cells, preserving them in a near-native state.
Cryo Dual-Beam FIB-SEM (e.g., Aquilos 2, Teneo) Integrated microscope for locating cells, depositing protection layers, and milling lamellae at cryo-temperatures.
Gas Injection System (GIS) for Pt/C Deposits a protective metallic layer on the ROI surface prior to milling, preventing ion damage to the biological material.
Cryo-Micromanipulator & Needles For physically lifting out the milled lamella and transferring it to a TEM grid.
Cryo AutoGrids or Half-Moon Grids Specialized TEM grids designed to securely hold FIB-lifted lamellae during transfer and TEM imaging.
Liquid Ethane/Propane Cooling System Creates the ultra-fast cooling medium necessary for vitrification of aqueous cellular samples.
Cryo Transfer Shuttles & Workstations Enable safe, cold, and contamination-free transfer of vitrified grids between storage, FIB, and TEM.
Specific Cell Culture Media & Viral Stocks For propagating host cells and generating infectious viral particles at appropriate titers for controlled MOI studies.
Fiducial Gold Beads (e.g., 10-15 nm colloidal gold) Often added before vitrification to serve as alignment markers during tomographic reconstruction in the TEM.

Cryo-Electron Tomography (cryo-ET) has become a pivotal technique for visualizing the structural dynamics of host-virus interactions in a near-native state. The core of cryo-ET data collection, tilt-series acquisition, is governed by a critical trade-off: achieving high-resolution 3D reconstructions while managing the total electron dose to preserve biological ultrastructure. This application note details protocols and strategies for optimizing tilt-series acquisition, specifically framed within research aimed at elucidating mechanisms of viral entry, replication, and assembly within host cells. Effective dose fractionation across a tilt-series is paramount for capturing high-fidelity snapshots of these transient interactions.

Quantitative Constraints in Tilt-Series Acquisition

The following tables summarize key technical parameters and their interdependencies.

Table 1: Primary Technical Constraints and Typical Values

Constraint Parameter Typical Range/Value Impact on Tilt-Series
Total Tolerable Dose 60-120 e⁻/Ų Limits total electrons per specimen area.
Angular Range ±60° to ±70° Defines the completeness of 3D reconstruction.
Angular Increment 1°-3° Finer increments improve resolution but increase dose or require dose fractionation.
Tilt Scheme Unidirectional, Bidirectional, Dose-Symmetric Affrades dose distribution and fidelity of features at different tilts.
Pixel Size at Detector 1-5 Å Defines Nyquist limit; smaller pixels increase dose for same SNR.
Defocus Range -3 µm to -8 µm Trade-off between phase contrast and CTF oscillations.
Ice Thickness < 100 nm (ideal: 50-80 nm) Thicker ice increases multiple scattering, reduces contrast & resolution.

Table 2: Dose Fractionation Schemes Comparison

Scheme Dose Distribution Advantages Disadvantages Best For
Linear (Unidirectional) Uniform across tilts Simple, fast, minimal stage movement. High dose on low-tilt, high-info views. Rapid screening, thick specimens.
Dose-Symmetric (Hagen et al.) Starts at 0°, alternates ± increments Maximizes info retention in low-tilt views. Complex stage movement, potential tracking errors. High-resolution subtomogram averaging, host-virus interfaces.
Saxton Scheme Dose weighted as 1/cos(tilt) Compensates for increased path length. Intermediate complexity. General purpose, improved z-resolution.
Bidirectional Two passes from ± max angle Reduces radiation-induced tilt. Total time, potential specimen change between passes. Very dose-sensitive specimens.

Detailed Experimental Protocols

Protocol 3.1: Cryo-ET Grid Preparation for Host-Virus Samples

  • Objective: To prepare a vitrified specimen of virus particles interacting with host cell membranes or organelles.
  • Materials: Purified virus, permissible host cell line (e.g., Vero, HEK293), cryo-EM grids (Quantifoil Au R2/2, 300 mesh), plunge freezer (e.g., Vitrobot Mark IV), 1% uranyl formate for negative stain validation.
  • Procedure:
    • Infection: Infect host cells at a high MOI (>10) for a defined time window corresponding to the interaction of interest (e.g., 30-60 min post-infection for entry).
    • Harvesting: Gently scrape or trypsinize cells. Pellet at low speed (500 x g, 5 min).
    • Vesicle Generation (Optional): Resuspend pellet in a hypotonic lysis buffer and mechanically disrupt to generate membrane vesicles with bound/virions. Clarify via low-speed centrifugation.
    • Grid Application: Apply 3-4 µL of sample to a glow-discharged grid. Blot for 3-6 seconds (blot force -5 to 0, 100% humidity) and plunge-freeze into liquid ethane.
    • Screening: Pre-screen grids using a 200kV cryo-microscope for ice quality, particle concentration, and presence of relevant complexes.

Protocol 3.2: Tilt-Series Acquisition with Dose-Symmetric Fractionation

  • Objective: To acquire a tilt-series optimized for high-resolution information transfer, suitable for subtomogram averaging of viral glycoproteins or host receptors.
  • Prerequisites: A 200-300kV FEG cryo-TEM with automated tomography package (e.g., SerialEM, Tomo5, UCSF Tomography).
  • Setup Parameters:
    • Microscope Conditions: 300kV acceleration voltage, energy filter slit width 20 eV, C2 aperture 70 µm, objective aperture removed.
    • Detector: Gatan K3 or Falcon 4 in counting mode.
    • Pixel Size: Aim for 1.0-1.5 Å/pixel at specimen level (calibrated magnification ~42,000-64,000x).
    • Target Defocus: -4.0 to -6.0 µm.
    • Total Dose: 80 e⁻/Ų (for a 50-80 nm thick ice area).
    • Tilt Range: -60° to +60°.
    • Increment: 3° for screening, 2° or 1.5° for high-quality data.
  • Acquisition Workflow in SerialEM:
    • Navigate & Focus: Find a region of interest with intact cellular features and multiple virus particles. Set eucentric height using the Wobbler.
    • Set Acquisition Parameters: Under the Tomography menu, select the Dose-Symmetric tilt order. Input starting angle (0°), increment (2°), and range (±60°).
    • Dose Fractionation: In the Low Dose panel, calculate the dose per frame. For 80 e⁻/Ų total dose over 61 tilts, dose/frame = ~1.31 e⁻/Ų. Set the exposure time accordingly.
    • Track and Acquire: Enable predictive tracking or cross-correlation tracking. Start the acquisition. The system will acquire in order: 0°, +1°, -1°, +2°, -2°, ... ±60°.
    • Save Data: Save the tilt-series as a stack of .mrc files with an associated .rawtlt or .tlt tilt angle file.

Visualizations

G Start Start: Aligned Cryo-TEM Grid P1 1. Locate Area of Interest (Ice <100nm, Virus-Cell Complex) Start->P1 P2 2. Set Eucentric Height (Wobbler Tool) P1->P2 P3 3. Configure Acquisition (300kV, Slit 20eV, Pixel 1.5Å) P2->P3 P4 4. Define Tilt Parameters: Range: ±60°, Increment: 2° P3->P4 P5 5. Select Dose-Symmetric Tilt Order (e.g., 0°, +2°, -2°...) P4->P5 P6 6. Calculate & Set Dose/Frame (Total 80 e⁻/Ų / N tilts) P5->P6 P7 7. Enable Predictive Real-Time Tracking P6->P7 P8 8. Acquire Tilt-Series P7->P8 P9 9. Save Stack + TLT File P8->P9 End Output: Tilt-Series Ready for Alignment & Reconstruction P9->End

Tilt-Series Acquisition Protocol Workflow

G TotalDose Total Budget (80 e⁻/Ų) Tilt0 0° Tilt (lowest path length, highest information) TotalDose->Tilt0 Higher dose/frame (~2-3x) TiltLow Low Tilts (±10-30°) (high information) TotalDose->TiltLow Moderate dose/frame TiltHigh High Tilts (±50-60°) (low information, long path) TotalDose->TiltHigh Lower dose/frame Outcome Optimized 3D Map: High-Fidelity Low-Tilt Info Preserved for Averaging Tilt0->Outcome TiltLow->Outcome TiltHigh->Outcome

Dose Fractionation Logic in Dose-Symmetric Scheme

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for Host-Virus Cryo-ET Sample Preparation

Item Function & Relevance to Host-Virus Studies
Quantifoil Au R2/2, 300 mesh grids Gold grids offer better thermal conductivity. R2/2 holey carbon provides large, clean ice areas for capturing extended cellular features.
Liquid Ethane Propane Mix (37%/63%) A superior cryogen for vitrification of thicker, cellular samples due to faster cooling rates than pure ethane, reducing ice crystal formation.
Fiducial Gold Beads (10-15 nm) Essential for post-acquisition tilt-series alignment. Protein A-coated beads can bind specifically to antibody-labeled samples.
Cytoskeleton Buffer (e.g., PHEM) A buffer for cell permeabilization/lysis that preserves microtubule and actin networks, crucial for studying virus trafficking.
Membrane Permeabilizers (Digitonin, Streptolysin O) For controlled cell permeabilization to allow entry of fiducials, labels, or to arrest viral processes at specific stages.
Cryo-ET Specific Negative Stain (1% UA in pH 4.5 water) For rapid grid screening to confirm presence of virus-host complexes before committing to cryo-EM time.
Graphene Oxide or Functionalized Graphene Support film to spread cell lysates, adsorb membranes, and provide a clean background, enhancing particle alignment.
JF646-HaloTag Ligand / Ni-NTA-Nanogold For correlated fluorescence microscopy and targeted labeling of specific host or viral proteins within cryo-lamellae.

Application Notes

Cryo-electron tomography (cryo-ET) is indispensable for visualizing the structural dynamics of host-virus interactions in a near-native state. The process of reconstructing a 3D tomogram from a series of 2D tilt images is computationally intensive and sensitive to noise and misalignment. Accurate alignment and back-projection are critical to resolving macromolecular complexes, such as viral fusion proteins engaging host cell receptors. Recent advances in algorithms and hardware acceleration have significantly improved the resolution and throughput of tomographic reconstruction, enabling the study of infection mechanisms at the molecular level. This directly informs antiviral drug design by identifying vulnerable stages in the viral life cycle.

Table 1: Comparative Analysis of Tomogram Reconstruction Software Packages (2023-2024)

Software Package Key Algorithm Alignment Method Denoising Integration Typical Resolution (Å) GPU Acceleration
IMOD WBP / SIRT Fiducial / Patch-based Non-Local Means 20-40 Limited (CUDA)
AreTomo SART Marker-free, Feature-based None 15-30 Yes (CUDA)
emClarity SIRT / MLEM Fiducial, Iterative Refinement Deep Learning (Cryo-CARE) 10-20 Yes (CUDA)
TomoPy Gridrec / SIRT Center-of-Rotation TV/Regularization 20-50 Yes (OpenCL)
M WBP Fiducial-based, Global Optimization External tools 20-40 No

Abbreviations: WBP (Weighted Back Projection), SIRT (Simultaneous Iterative Reconstruction Technique), SART (Simultaneous Algebraic Reconstruction Technique), MLEM (Maximum Likelihood Expectation Maximization). Resolution range is for cellular cryo-ET of host-virus samples.

Experimental Protocols

Protocol 2.1: Fiducial-Based Tilt-Series Alignment and Reconstruction using IMOD

Objective: To generate a preliminary 3D tomogram from a cryo-ET tilt series using fiducial markers for high-precision alignment.

Materials:

  • Tilt-series data (e.g., .mrc stack)
  • IMOD software package (v4.12+)
  • Computationally equipped workstation (≥64 GB RAM, GPU recommended)

Procedure:

  • Preprocessing: Use framealign or motioncor2 for dose-weighted frame alignment and integration. Generate a dose filter with ctfplotter.
  • Coarse Alignment: In etomo, create a new project. Load the stack and set basic parameters (pixel size, tilt angles). Run the initial coarse alignment to correct for large shifts and rotations.
  • Fiducial Model Creation: Use the Fiducial Model function. Manually place gold bead markers (≥10) on at least 3 frames (e.g., 0°, ±45°). Run Auto-Tracking to track beads through the series.
  • Fine Alignment: Execute the Fiducial Fine Alignment function. Inspect the residual error report. Accept if total error is < 1.5 nm. Use the Alignment Parameters tool to view and potentially exclude high-error beads.
  • Tomogram Generation: Proceed to the Tomogram Positioning step. Define the volume of interest and thickness. Select Weighted Back-Projection (WBP) for speed or SIRT (15-20 iterations) for better noise suppression. Generate the tomogram.
  • Output: The final tomogram (e.g., tomogram.rec) is ready for denoising or segmentation.

Protocol 2.2: Marker-Free Alignment and Denoising with AreTomo and IsoNet

Objective: To reconstruct and denoise a tomogram without fiducial markers, leveraging deep learning for improved signal-to-noise ratio.

Materials:

  • Motion-corrected tilt series (.mrc)
  • AreTomo (v1.3+)
  • IsoNet software package
  • GPU workstation (NVIDIA, ≥12 GB VRAM)

Procedure:

  • Marker-Free Alignment in AreTomo:
    • Launch AreTomo in command line or GUI mode.
    • Input the tilt series and angle file. Set PixelSize and TiltAxis.
    • Set Algo to 1 (patch tracking). Enable FlipVol and OutImod if using IMOD for post-processing.
    • Run alignment. Check the output *.log file for alignment error metrics. A successful run typically shows a mean residual error below 2 pixels.
    • Output the aligned stack and tilt file (.aln).
  • Reconstruction: In AreTomo, use the --Reconstruction flag. Choose the SART algorithm with 5-10 iterations and --Bin 2 for a binned reconstruction to speed up subsequent denoising.
  • Deep Learning Denoising with IsoNet:
    • Prepare the binned tomogram and its projection images (use tom2pred or AreTomo's projection function).
    • Run isonet.py correct with the provided tomogram and projections. Use default parameters for a first pass (--gpuID 0, --split_method consecutive).
    • IsoNet will train a model to predict missing-wedge corrected and denoised volumes. Training typically requires 50-100 epochs.
    • The final output is a denoised, missing-wedge corrected tomogram (denoised.mrc) suitable for high-fidelity segmentation of viral and host structures.

Diagrams

G node1 Raw Tilt Series Collection node2 Preprocessing: Motion Correction & CTF Estimation node1->node2 node3 Alignment node2->node3 node3a Fiducial-Based (e.g., IMOD) node3->node3a node3b Marker-Free (e.g., AreTomo) node3->node3b node4 3D Reconstruction (WBP, SIRT, SART) node3a->node4 node3b->node4 node5 Raw Tomogram node4->node5 node6 Denoising node5->node6 node6a Traditional (NLM, TV) node6->node6a node6b Deep Learning (IsoNet, CryoCARE) node6->node6b node7 Final Denoised Tomogram node6a->node7 node6b->node7 node8 Analysis: Segmentation & Interpretation node7->node8

Tomogram Reconstruction and Denoising Workflow

G cluster_path Key Host-Virus Interaction Imaging Targets V Viral Particle S1 1. Attachment: Viral Spike (S) & Host Receptor V->S1 H Host Cell Membrane H->S1 S2 2. Membrane Fusion / Penetration S1->S2 S3 3. Genome Release & Replication Complexes S2->S3 S4 4. Viral Assembly & Budding Sites S3->S4

Cryo-ET Targets in Host-Virus Research

The Scientist's Toolkit

Table 2: Essential Research Reagent Solutions for Cryo-ET Sample Preparation

Item Function & Rationale Example Product / Specification
Quantifoil R2/2 Au Cryo-EM grids with a regular hole pattern and a gold support film. Gold provides better conductivity and thermal stability than copper, reducing beam-induced motion. Quantifoil Au 300 mesh, R2/2 (2µm holes)
10nm Colloidal Gold Fiducial markers for high-precision tilt-series alignment. Uniform size is critical for accurate tracking by software. BSA-treated 10nm Gold Particles (Aurion or Cytodiagnostics)
Gradual Freeze Device Enables controlled, blot-free vitrification of sensitive samples like infected cells, preserving membrane integrity and preventing ice crystal formation. EM GP2 (Leica) or VitroJet (Thermo Fisher)
FIB-SEM Mill Prepares thin lamellae (100-300nm) from vitrified infected cells for in-situ cryo-ET, allowing imaging of interior structures. Aquilos 2 or Crossbeam 550 (Zeiss)
Plasma Cleaner Hydrophilizes the grid surface immediately before application of the sample, ensuring even distribution and appropriate ice thickness. Gatan Solarus or Tergeo-EM (Pie Scientific)
Anti-Curling Solution A compound like bacitracin used during blotting to promote a more uniform ice layer and prevent grid curling, especially for large cellular samples. 0.1% bacitracin in sample buffer

This Application Note provides detailed protocols for the segmentation and visualization of key structural components—specifically viruses, organelles, and host proteins—within cryo-electron tomography (cryo-ET) data. The procedures are framed within a broader thesis on utilizing cryo-ET to elucidate host-virus interaction mechanisms, aiming to provide actionable methodologies for researchers and drug development professionals.

Key Research Reagent Solutions

The following table details essential materials and their functions for cryo-ET workflow focusing on host-virus samples.

Item Name Function/Brief Explanation
Vitrification System (e.g., Vitrobot) Rapidly plunges the hydrated biological sample into liquid ethane to form amorphous ice, preserving native-state structures.
Fiducial Gold Markers (10-15 nm) Provide reference points for accurate alignment of tilt series during tomogram reconstruction.
Cryo-Focused Ion Beam (Cryo-FIB) Mill Thins vitrified cellular samples (lamella preparation) to electron transparency (~200 nm) for TEM imaging.
300 keV Cryo-Transmission Electron Microscope Provides high-resolution imaging of vitrified samples with minimal radiation damage.
Direct Electron Detector Captures high-contrast, low-noise images from tilt series, crucial for high-fidelity reconstruction.
Subtomogram Averaging Software (e.g., RELION, EMAN2) Aligns and averages repeating structures (e.g., viral spikes, ribosomes) to achieve high-resolution details.
Cellular Tomography Software (e.g., IMOD, SerialEM) Acquires tilt series images and reconstructs them into a 3D tomogram.
Segmentation Software (e.g., Amira, ChimeraX) Manually or semi-automatically delineates structures of interest within the 3D tomogram volume.

Protocols

Protocol 1: Cryo-FIB Milling for Host-Cell Lamella Preparation

Objective: To produce an electron-transparent lamella from virus-infected cells for cryo-ET imaging. Materials: Vitrified infected cells on EM grid, Cryo-FIB/SEM microscope, sputter coater. Procedure:

  • Transfer: Load the vitrified grid into the Cryo-FIB/SEM microscope shuttle under liquid nitrogen conditions.
  • Sputter Coat: Apply a thin layer of platinum (~5-10 nm) to the grid surface to enhance conductivity and protect the sample.
  • Rough Milling: Using a high ion current (e.g., 1 nA), mill large trenches on either side of the target cell region.
  • Fine Milling & Polishing: Gradually reduce the ion current (to ~100 pA) to thin the lamella to the target thickness of 200-300 nm. Use SEM imaging to monitor progress.
  • Final Inspection: Acquire a low-dose SEM image to confirm lamella quality and uniformity.
  • Transfer for TEM: Under cryogenic conditions, transfer the shuttle to the cryo-TEM for tilt series acquisition.

Protocol 2: Tilt Series Acquisition and Tomogram Reconstruction

Objective: To collect a tilt series and reconstruct a 3D tomogram of the infected cellular lamella. Materials: Cryo-FIB lamella, 300 keV Cryo-TEM with a Direct Electron Detector, tomography acquisition software (e.g., SerialEM). Procedure:

  • Grid Loading: Insert the shuttle with the lamella into the cryo-TEM stage, maintaining temperature below -170°C.
  • Site Selection: At low magnification, identify a region of interest (ROI) containing cell features and potential viruses.
  • Fiducial Tracking: Acquire a zero-tilt image. Align the stage to ensure fiducial markers are in the field of view throughout the tilt range.
  • Acquisition Setup: Set a dose-symmetric tilt scheme from -60° to +60° with a 2-3° increment. Set a total cumulative dose of 100-150 e⁻/Ų.
  • Automated Acquisition: Initiate the automated tilt series acquisition. The software will track and correct for image shift and focus.
  • Alignment & Reconstruction: Using IMOD software, align the tilt series images based on fiducial gold markers. Reconstruct the volume using a weighted back-projection or SIRT algorithm to generate the tomogram (.rec file).

Protocol 3: Semi-Automated Segmentation of Viruses and Organelles

Objective: To identify and label viral particles, mitochondria, and endoplasmic reticulum within the reconstructed tomogram. Materials: Reconstructed tomogram, segmentation software (e.g., Amira, ORS Dragonfly). Procedure:

  • Tomogram Import & Denoising: Import the .rec or .mrc file. Apply a denoising filter (e.g., Non-Local Means or Deep Learning-based denoiser like Topaz) to enhance features.
  • Template Matching (Viruses): For known, repetitive viral structures (e.g., capsids), use a template matching function (e.g., in EMAN2) to locate particles across the volume automatically.
  • Thresholding & Seeding (Organelles):
    • For lipid membranes (mitochondria, ER), apply a global threshold based on voxel intensity to highlight membranous features.
    • Use the "Magic Wand" or "Interactive Thresholding" tool to select connected regions belonging to a single organelle.
  • Label Field Creation: Create separate label fields for "Viral Capsids," "Mitochondria," and "ER."
  • Morphological Operations: Apply "Closing" and "Opening" operations to smooth the boundaries of segmented objects and fill small holes.
  • Surface Rendering: Generate a 3D mesh surface from each label field for visualization.

Protocol 4: Subtomogram Averaging of Host Protein Complexes

Objective: To achieve high-resolution structure of non-repetitive host protein complexes (e.g., ribosomes, inflammasomes) by averaging. Materials: Tomogram, particle coordinates, subtomogram averaging software (e.g., RELION-4.0, M). Procedure:

  • Particle Picking: Manually or semi-automatically pick positions of protein complexes of interest from the denoised tomogram.
  • Extraction: Extract sub-volumes (e.g., 64x64x64 voxels) centered on each particle coordinate.
  • Initial Reference Generation: Create an initial low-resolution reference, either from a previous similar structure or by averaging all extracted particles after rough alignment.
  • Iterative Alignment & Averaging: Run multiple cycles of iterative alignment (rotational and translational) of each sub-volume to the reference, followed by averaging to create a new, higher-resolution reference.
  • Classification (Optional): Perform 3D classification to separate heterogeneous conformational states of the complex.
  • Resolution Assessment: Calculate the Fourier Shell Correlation (FSC) between two independently refined half-maps. Report the resolution at FSC=0.143.

Data Presentation: Quantitative Segmentation Metrics

The following table summarizes typical quantitative outputs from the segmentation and analysis of a tomogram of a virus-infected cell.

Segmented Component Typical Count per Tomogram Average Volume (nm³) Key Co-localization Metric
Viral Particles (e.g., HSV-1) 5 - 20 5.2 x 10⁵ 85% within 50 nm of ER membrane
Mitochondria 3 - 8 1.8 x 10⁷ 60% show altered morphology
Endoplasmic Reticulum 1 - 3 (contiguous network) N/A 95% of viral particles associated
Host Ribosomes 500 - 2000 4.5 x 10³ 30% decrease in polysome clusters
Viral Glycoprotein Spikes 200 - 600 (per virion) ~80 (per spike) N/A

Diagrams

Diagram 1: Cryo-ET Host-Virus Analysis Workflow

G A Cell Culture & Virus Infection B Vitrification (Plunge Freezing) A->B C Cryo-FIB Milling (Lamella Preparation) B->C D Cryo-TEM Tilt Series Acquisition C->D E Tomogram Reconstruction D->E F Segmentation & 3D Visualization E->F G Subtomogram Averaging F->G H Quantitative Analysis & Modeling G->H

Diagram 2: Segmentation Decision Logic for Tomogram Features

G Start Start with Tomogram Slice Q1 Spherical/ Polygonal Shape? (20-200 nm) Start->Q1 Q2 Tubular/Double Membrane? Q1->Q2 No V Label as VIRUS Q1->V Yes Q3 Reticulated Network? Q2->Q3 No M Label as MITOCHONDRION Q2->M Yes Q4 Small, Dense, Repetitive? Q3->Q4 No ER Label as ENDOPLASMIC RETICULUM Q3->ER Yes RP Label as RIBOSOME/ PROTEIN COMPLEX Q4->RP Yes U Further Analysis Required Q4->U No

This protocol details the application of sub-tomogram averaging (STA) within a broader cryo-electron tomography (cryo-ET) research program focused on elucidating host-virus interaction mechanisms. The goal is to derive high-resolution in-situ structures of viral protein complexes, fusion machinery, and host-cell receptors from tomographic data of vitrified infected cells. This structural information is critical for identifying vulnerabilities in the viral life cycle and informing targeted therapeutic intervention.

Table 1: Resolution Determinants in Sub-tomogram Averaging

Factor Typical Range/Value Impact on Final Resolution Notes for Host-Virus Studies
Tilt Series Defocus -3 µm to -8 µm Crucial for initial contrast; errors degrade resolution. Consistent defocus aids alignment of heterogeneous complexes.
Pixel Size at Detector 1.0 – 3.5 Å Defines Nyquist limit. Finer sampling (<2.0 Å) needed for small antiviral drug targets.
Total Electron Dose 60 – 150 e⁻/Ų Higher dose improves SNR but increases radiation damage. Lower dose (80-100 e⁻/Ų) preserves delicate host-virus interfaces.
Number of Subtomograms 1,000 – 100,000+ Increases linearly with resolution (^3 dependency). Viral surface spikes often require >10,000 particles.
Initial Angular Accuracy >15° error Major bottleneck for high-resolution refinement. Initial model from known viral structure often used.
B-Factor (Temperature Factor) 50 – 2000 Ų Higher values indicate faster signal fall-off. Host membrane environments often exhibit higher B-factors.

Table 2: Typical STA Workflow Output Metrics

Processing Stage Key Metric Target Value (for ~3Å goal) Quality Check
Tomogram Reconstruction SNR (Cryo-CARE) >0.8 Denoising applied post-reconstruction.
Particle Picking False Positive Rate <20% Manual validation on slice views.
Initial Alignment Cross-Correlation Score >0.4 Indicates reasonable initial orientation.
3D Classification Class Heterogeneity Clear separation Identifies distinct conformational states.
Final Refinement FSC 0.143 Threshold ≤3.0 Å Gold-standard, mask-corrected.
Local Resolution Variation across map 2.8 – 5.0 Å Core viral protein typically highest res.

Detailed Experimental Protocol: STA for Viral Envelope Glycoproteins

Sample Preparation & Tilt Series Acquisition

  • Material: Vitrified cell monolayer on EM grid infected with virus (MOI ~5-10).
  • Protocol:
    • Lamella Preparation: Using a focused ion beam/scanning electron microscope (FIB/SEM), mill 150-200 nm thick lamellae from regions of infected cells. Polish at low keV (2-5 kV) to reduce amorphous layer.
    • Screening: Identify lamellae with intact cell morphology and visible viral particles near the surface using low-dose TEM.
    • Tilt Series Acquisition: Acquire a dose-symmetric tilt series from -60° to +60° with a 2° or 3° increment using a 300 kV cryo-TEM equipped with a direct electron detector and energy filter (slit width 20 eV).
    • Dose Fractionation: Total cumulative dose target: 80-100 e⁻/Ų. Use dose compensation (e.g., SerialEM or Tomography 5 software). Frame-based acquisition recommended.
    • Metadata Export: Save *.mdoc file with alignment parameters, defocus, and dose per tilt.

Tomogram Reconstruction & Denoising

  • Software: IMOD, AreTomo, or Warp.
  • Protocol:
    • Frame Alignment & Motion Correction: Use MotionCor2 or Warp on dose-fractionated frames per tilt.
    • Coarse Alignment: Generate initial aligned stack using fiducial markers (e.g., 10nm gold beads) or patch-tracking.
    • Refined Alignment & CTF Correction: Apply CTFFIND-4.1 or Gctf per tilt. Perform refined alignment incorporating CTF parameters.
    • Reconstruction: Compute a 3D reconstruction using weighted back-projection (WBP) or simultaneous iterative reconstruction technique (SIRT) with 4-10 iterations (bin 2-4 initially).
    • Denoising: Apply deep learning-based denoising (Cryo-CARE, Topaz-Denoise) using matched cryo-tomogram pairs to enhance SNR while preserving structural details.

Sub-tomogram Extraction & Averaging

  • Software: Dynamo, RELION, EMAN2, or AV3.
  • Detailed Protocol (Dynamo-centric):
    • Template Creation: Generate an initial low-pass filtered (40-60 Å) reference from a known homologous atomic model or a crude average from manually picked particles.
    • Particle Picking:
      • Manual: Annotate viral membrane or glycoprotein densities in binned tomograms.
      • Template Matching: Use low-pass filtered reference to scan tomogram. Set cross-correlation threshold to include top 5-10% peaks.
      • Curate: Manually remove false positives from host membranes or irrelevant densities.
    • Particle Table Creation: Create a Dynamo table containing coordinates (x, y, z), and initial Euler angles (if known). Extract sub-volumes with a box size ~1.5x particle diameter.
    • Initial Alignment & Averaging:
      • Run an initial alignment project with a broad angular search, limited shifts, and the initial template.
      • Apply CTF correction during alignment (phase-flip).
      • Generate first average; low-pass filter to the estimated resolution.
    • 3D Classification: Use PCA-based classification (`dynamo_classification) into 3-5 classes to separate conformational states, symmetry mismatches, or false picks.
    • Iterative Refinement:
      • Refine selected class(es) using progressively tighter angular and shift searches.
      • Impose symmetry (e.g., C3 for trimeric spike) if known and validated.
      • Monitor Fourier Shell Correlation (FSC) between two independently refined half-maps. Apply a soft mask around the particle for FSC calculation.
      • Run multiple cycles until FSC convergence.
    • Sharpening & Visualization: Apply post-processing (B-factor sharpening) using RELION's postprocess or dynamo_map_sharpen. Visualize in ChimeraX or UCSF Chimera.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for STA in Host-Virus Research

Item Function Example/Product Application Note
Quantifoil R2/2 Au 300 EM grid with holey carbon film. Provides support for cellular growth and lamella milling. Au grids preferred for FIB-SEM conductivity.
FIB-SEM Micrometer-Sized Beads Fiducials for lamella milling. e.g., 100nm Polystyrene Beads Sprayed on grid to locate milling region.
10-15nm Colloidal Gold Fiducials for tilt series alignment. Aurion or BBI Solutions Conjugated with BSA for even distribution on lamella.
Plasma FIB (PFIB) Source (Xe) Faster, cleaner milling of biological lamellae. Thermo Fisher Scientific Helios Hydra Reduces Ga+ ion implantation damage.
Direct Electron Detector High DOE, fast readout for dose fractionation. Gatan K3, Falcon 4 Essential for high-resolution STA.
Energy Filter Zero-loss energy filtering. Gatan GIF BioQuantum Improves SNR by removing inelastic electrons.
Cryo-TEM Stage Stable, auto-loading stage for unattended data collection. Thermo Fisher Scientific Autoloader Enables high-throughput tilt series collection.

Visualization Diagrams

STA_Workflow START Infected Cell Sample (Vitrified) TS_ACQ Tilt Series Acquisition START->TS_ACQ FIB Milling TOMO_REC Tomogram Reconstruction & Denoising TS_ACQ->TOMO_REC Align & Back-Project PICK Sub-tomogram Picking & Extraction TOMO_REC->PICK Template/Mannual Pick ALIGN Alignment, Classification & Iterative Refinement PICK->ALIGN Initial Model HIGH_RES High-Resolution 3D Map & Analysis ALIGN->HIGH_RES FSC Validation DRUG_DESIGN Molecular Analysis & Drug Design Insights HIGH_RES->DRUG_DESIGN Atomic Modeling

Title: Sub-tomogram Averaging Workflow from Sample to Map

HostVirus_STA_Pathway VIRUS Viral Particle (Tomogram Density) SPIKE Viral Envelope Glycoprotein (Spike) VIRUS->SPIKE RECEPTOR Host Cell Surface Receptor SPIKE->RECEPTOR Binds FUSION Membrane Fusion Machinery SPIKE->FUSION Triggers STA_PROC STA Processing (Extract, Align, Average) SPIKE->STA_PROC Sub-volumes RECEPTOR->STA_PROC Sub-volumes FUSION->STA_PROC Sub-volumes ANTIBODY Neutralizing Antibody Fab ANTIBODY->SPIKE Blocks ANTIBODY->STA_PROC Sub-volumes MAP High-Res In-Situ Complex Map STA_PROC->MAP OUTPUT1 Identify Vulnerable Sites MAP->OUTPUT1 OUTPUT2 Guide Antibody Design MAP->OUTPUT2 OUTPUT3 Rational Drug Design MAP->OUTPUT3

Title: STA Informs Drug Design via Host-Virus Structures

Application Note: Structural Insights into Viral Entry and Assembly via Cryo-ET

Cryo-Electron Tomography (Cryo-ET) has revolutionized our understanding of host-virus interactions by visualizing macromolecular complexes in their native, cellular context. This note details landmark findings enabled by this technology.

HIV-1: Envelope Glycoprotein Dynamics and Maturation

Cryo-ET revealed the structural conformation of native HIV-1 envelope glycoprotein (Env) trimers on the virion surface and the dramatic structural maturation of the Gag polyprotein lattice.

  • Key Finding: Native Env trimers are inherently flexible and exist in both "closed" and "open" conformations, informing immunogen design.
  • Key Finding: Immature virions show a spherical Gag lattice; upon proteolytic cleavage during maturation, it reorganizes into a conical, hexagonal lattice of CA protein, essential for infectivity.

SARS-CoV-2: Spike Protein Conformations and Membrane Fusion

In situ Cryo-ET visualized the full-length SARS-CoV-2 spike (S) glycoprotein on the virion and captured its fusion machinery in action.

  • Key Finding: S protein exists predominantly in a prefusion state on the virion, with a subset in the postfusion state. Subtomogram averaging provided atomic models of both states.
  • Key Finding: Imaging of infected cells captured double-membrane vesicles (DMVs), sites of viral replication, and the moment of membrane fusion, revealing pore formation.

Herpesviruses: Nuclear Egress and Tegument Organization

Cryo-ET elucidated the complex process of herpes simplex virus (HSV) nuclear egress and the asymmetric architecture of the cytomegalovirus (HCMV) tegument.

  • Key Finding: Visualization of nucleocapsids traversing the nuclear envelope via the nuclear egress complex (NEC), which transiently deforms the inner and outer nuclear membranes.
  • Key Finding: HCMV virions exhibit an asymmetric, protein-dense tegument layer between capsid and envelope, organized into specific subdomains with distinct protein compositions.

Table 1: Quantitative Landmarks from Cryo-ET Studies

Virus Resolved Feature Key Quantitative Measurement Biological Implication
HIV-1 Native Env trimer spacing ~14-16 nm between trimers on virion surface Sparse distribution informs antibody accessibility.
HIV-1 Gag lattice curvature (immature) Radius of curvature: ~35-40 nm Defines the assembly scaffold for the nascent virion.
SARS-CoV-2 Prefusion S protein density ~25 Å resolution from subtomogram averaging Enabled structure-guided vaccine and therapeutic design.
SARS-CoV-2 Double-membrane vesicle (DMV) size Diameter: ~200-300 nm Identified primary site for viral RNA synthesis.
HSV-1 Nuclear egress complex lumen Diameter: ~15-20 nm Constrained size for capsid deformation during transit.
HCMV Tegument thickness Variable, from ~30 nm to >70 nm Reflects complex, organized protein layering essential for infectivity.

Protocol: In Situ Cryo-ET Workflow for Imaging Viral Entry

Objective: To capture the structural events of SARS-CoV-2 spike-mediated membrane fusion in host cells.

I. Sample Preparation

  • Cell Culture & Infection: Seed Vero E6 or susceptible cells expressing ACE2 onto glow-discharged, gold-coated EM grids. Infect with SARS-CoV-2 (proper biosafety level-3 protocols required) at low MOI (~0.1-1) for a short duration (e.g., 5-15 mins for early entry events).
  • Vitrification: At desired time point, plunge-freeze the grid in liquid ethane using a vitrification robot (e.g., Vitrobot). Maintain >95% humidity.
  • Fluorescent Screening (Optional): Use cryo-correlated light and electron microscopy (cryo-CLEM) to identify cells expressing a fluorescently tagged viral component.

II. Cryo-ET Data Acquisition

  • Grid Loading: Transfer the vitrified grid to a 300 keV cryo-transmission electron microscope equipped with a biasing holder and a direct electron detector.
  • Low-Dose Tilt-Series Acquisition: Using serialEM or equivalent software, acquire a tilt-series from -60° to +60° with a 2-3° increment at a nominal magnification of 26,000x (pixel size ~5.3 Å). Use a cumulative dose of <100 e⁻/Ų.
  • Fiducial-less Tracking: Employ patch-tracking or feature-based tracking for image alignment during tilt.

III. Tomogram Reconstruction & Analysis

  • Reconstruction: Align tilt-series using IMOD or similar. Reconstruct tomogram via weighted back-projection or SIRT-like algorithms.
  • Subtomogram Averaging (For S Protein):
    • Particle Picking: Manually or semi-automatically pick coordinates of spike densities from the tomogram.
    • Alignment & Averaging: Use PyTom, RELION, or M to align and average thousands of subvolumes. Iteratively refine angles and positions.
    • Classification: Perform 3D classification to separate pre-fusion, intermediate, and post-fusion states.
  • Segmentation & Visualization: Manually or using machine learning (e.g., EMAN2, TomoSegMemTV) segment membranes, viral capsids, and spike densities. Render 3D models in ChimeraX.

G A Seed Cells on EM Grid B Infect with Virus (Low MOI) A->B C Vitrification (Plunge-Freeze) B->C D Cryo-Fluorescence Screening (Optional) C->D E Cryo-ET Tilt-Series Acquisition C->E D->E F Tomogram Reconstruction E->F G Subtomogram Averaging & Analysis F->G H 3D Segmentation & Model Building G->H

Title: Cryo-ET Workflow for Viral Entry Imaging

H cluster_host Host Cell Membrane cluster_virus SARS-CoV-2 Virion ACE2 ACE2 Receptor State1 1. Prefusion (Virion Attached) Memb Lipid Bilayer S1 S1 Subunit (RBD) S2 S2 Subunit (Fusion) Memv Viral Envelope State1->ACE2  RBD Binding State2 2. Postfusion (Pore Formation) State1->State2  Proteolytic  Cleavage &  Conformational Change State2->Memb  Fusion  Pore

Title: SARS-CoV-2 Spike-Mediated Membrane Fusion

The Scientist's Toolkit: Key Reagents for Host-Virus Cryo-ET

Table 2: Essential Research Reagent Solutions

Reagent / Material Function in Cryo-ET of Viruses Example / Specification
Quantifoil or C-flat EM Grids Provide a thin, stable carbon support film over holes for cellular imaging. Au 300 mesh, R 2/1 or R 1.2/1.3; often glow-discharged for hydrophilicity.
Cryo-Protectant (Optional) Can improve vitrification thickness for thicker cellular samples. 10-20% (v/v) glycerol, trehalose, or FBS in culture medium.
Fiducial Gold Beads High-contrast markers for aligning tilt-series images. Protein A-conjugated 10-15 nm colloidal gold.
Focused Ion Beam (FIB) Mill Thins vitrified cellular samples (lamella preparation) for electron transparency. Aquilos 2 or SEM/FIB dual-beam system with cryo-stage.
Direct Electron Detector Captures high-resolution images with high sensitivity at low electron doses. Gatan K3 or Falcon 4 camera in electron counting mode.
Correlative Markers For cryo-CLEM; allow fluorescence targeting of infection sites. TetraSpeck beads or fluorescent dye (e.g., CellMask) stains.
Image Processing Software Suite For tilt-series alignment, reconstruction, segmentation, and averaging. IMOD, Warp, RELION, EMAN2, PyTom, ChimeraX.
BSL-3 Facility (for pathogens) Mandatory for safe culture and preparation of viruses like SARS-CoV-2 and HIV. Certified containment lab with protocols for inactivating samples pre-EM.

Navigating the Ice: Common Cryo-ET Challenges and Proven Optimization Strategies

Within cryo-electron tomography (cryo-ET) studies of host-virus interactions, the integrity of the vitrified specimen is paramount. Ice contamination (crystalline ice, frost) and sample devitrification (the conversion of amorphous vitreous ice into crystalline ice) are primary technical obstacles. They degrade resolution, introduce artifacts, and can render data unusable. These challenges are acute when studying delicate, dynamic interfaces between host cellular structures and viral particles. This document outlines best practices for grid handling to preserve optimal ice quality from vitrification through data collection.

Understanding the Adversaries: Contamination and Devitrification

  • Ice Contamination: Occurs when water vapor condenses and freezes onto the grid surface. Sources include ambient humidity, breath, and temperature gradients.
  • Devitrification: The reorganization of water molecules from a vitrified (amorphous, glass-like) state to a crystalline state. It is primarily temperature-dependent, occurring most rapidly in the range of -137°C to -150°C (the "devitrification zone").

Table 1: Common Artifacts, Causes, and Consequences

Artifact Type Primary Cause Consequence for Host-Virus Imaging
Hexagonal Crystalline Ice Poor blotting, slow freezing Obscures macromolecular details, causes scattering.
Frost/Ice Layers on Grid Humidity during transfer/loading Creates uneven ice, prevents accurate targeting.
Sample Devitrification Warming above -150°C for prolonged periods Loss of high-resolution information, bubbling.
Ethane Contamination Improper blotting after plunging Introduces crystalline structures, alters contrast.

Critical Temperature Parameters

Successful vitrification and storage require rapid transition through dangerous temperature zones.

Table 2: Key Temperature Benchmarks for Vitreous Ice Stability

Temperature Zone Range Risk & Requirement
Devitrification Zone -137°C to -150°C HIGH RISK. Minimize time in this range.
Safe Storage Below -150°C (Ideally < -170°C) LONG-TERM STABILITY. No devitrification.
Liquid Nitrogen (LN₂) -196°C Safe for storage, but prone to condensation during handling.
Plunging Coolant Ethane/Propane mix at LN₂ temp ~ -182°C for rapid, uniform vitrification.

Protocols for Grid Handling

Pre-Vitrification Grid Preparation

Objective: Ensure grids are clean, hydrophilic, and free of contaminants.

  • Glow Discharge: Use a glow discharger at medium power for 30-60 seconds. Create hydrophilic surface for even sample adhesion. Use just prior to application of sample.
  • Grid Storage: Store unused grids in a clean, dry environment (desiccator).

Vitrification Protocol (Manual Plunge Freezing)

Materials: Vitrification device, forceps, humidity controller (< 40% RH), blotting paper, ethane/propane mix, LN₂, grid boxes.

  • Environmental Control: Reduce lab humidity to < 40% to minimize frost.
  • Coolant Preparation: Condense ethane/propane mix in a small metal cup submerged in LN₂ until slushy.
  • Sample Application: Apply 3-5 µL of sample to the glow-discharged grid. Wait 5-60 seconds for adsorption (optimize for each sample).
  • Blotting: Gently blot from the back side of the grid for 2-5 seconds to achieve an optimal, thin ice layer. Critical: Avoid over-blotting (thick ice) or under-blotting (crystalline ice).
  • Plunging: Rapidly plunge the grid into the ethane/propane slush at a consistent speed. Hold submerged for > 5 seconds.
  • Transfer to Storage: Under LN₂, quickly transfer the grid from the ethane cup to a pre-cooled grid box submerged in LN₂. Ensure the grid box is never warmed above -150°C.

Grid Transfer and Storage

Objective: Maintain grids below -150°C during all transfers.

  • LN₂-Cooled Tools: Always use pre-cooled forceps, grid boxes, and transfer stations.
  • Use of Workstation: Perform transfers in a humidified glove box or under a continuous LN₂ vapor stream to prevent atmospheric moisture condensation.
  • Long-Term Storage: Store grid boxes in LN₂ dewars with robust inventory systems. Avoid storing in the vapor phase of auto-filling dewars where temperature fluctuations can occur.

Loading into the Cryo-Electron Microscope

Objective: Transfer the grid to the microscope column without warming or frosting.

  • Pre-Cool the Holder: Ensure the cryo-holder is properly cooled and decontaminated (frost-free) before loading.
  • Rapid Transfer: Use a cryo-transfer station or shuttle. The grid should be exposed to air for less than 3-5 seconds during the loading process.
  • In-Scope Dewar Fill: Fill the microscope side-entry dewar with LN₂ well in advance to ensure stable, cold conditions before inserting the holder.

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Cryo-ET Grid Handling

Item Function & Importance
Quantifoil or C-Flat Grids Holey carbon films providing thin, stable vitreous ice over holes for imaging.
Ethane/Propane Mix Cooling agent with higher heat capacity than pure ethane, enabling faster vitrification rates.
Blotting Paper (Filter Paper) For removing excess sample to form a thin film; variable grade and porosity affect blotting.
Glow Discharger Creates a hydrophilic grid surface for even sample spreading and adhesion.
Cryo-Grid Storage Box LN₂-compatible box for safe, organized storage of multiple grids.
Autogrid or Clip Rings Specialized carriers for specific microscope stages (e.g., Thermo Fisher Autogrid).
Anti-Capillary Tweezers Prevent liquid suction between tips, crucial for clean blotting and handling.
Cryo-Transfer Station/Shuttle Maintains a cold, dry environment during grid transfer to the microscope.
Humidity/Temperature Monitor Ensures ambient conditions are optimized during sample application and blotting.

Visualizing Workflows and Relationships

G Start Start: Sample & Grid Prep Vit Vitrification Process Start->Vit HD High Humidity (>60%) Start->HD leads to LD Low Humidity (<40%) Start->LD leads to Store Storage & Transfer Vit->Store Slow Slow Freezing or Warm Temp Vit->Slow leads to Fast Rapid Plunge in Ethane/Propane Vit->Fast leads to Scope Microscope Imaging Store->Scope WarmX Warm Transfer (> -150°C) Store->WarmX leads to ColdX Cold Transfer (< -170°C) Store->ColdX leads to BadIce ✗ Crystalline Ice or Frost HD->BadIce GoodIce Thin, Vitreous Ice LD->GoodIce Slow->BadIce Fast->GoodIce WarmX->BadIce ColdX->GoodIce Success High-Quality Cryo-ET Data GoodIce->Success Fail Poor Data Artifacts BadIce->Fail

Grid Handling Impact on Ice Quality

G Title Cryo-ET Workflow for Host-Virus Studies with Critical Ice Quality Checkpoints CP1 Checkpoint 1: Pre-Vitrification Title->CP1 S1 Culture/Infect Cells Purify Virions Prepare Lamellae CP1->S1 CP2 Checkpoint 2: Vitrification S2 Glow Discharge Blot & Plunge CP2->S2 CP3 Checkpoint 3: Storage/Transfer S3 LN₂ Transfer & Long-Term Storage CP3->S3 CP4 Checkpoint 4: Microscope Loading S4 Grid Screening Tomogram Acquisition CP4->S4 QC1 Q: Humidity <40%? Grid Hydrophilic? S1->QC1 QC2 Q: Blotting Time Optimal? Plunge Speed Fast? S2->QC2 QC3 Q: Temp Always < -170°C? No Frost Visible? S3->QC3 QC1->CP2 QC2->CP3 QC3->CP4 QC4 Q: Holder Frost-Free? Transfer < 5 sec?

Cryo-ET Workflow with Ice Quality Checkpoints

Understanding the structural dynamics of host-virus interactions at the molecular level is a central goal of modern virology and drug development. Cryo-electron tomography (cryo-ET) is uniquely positioned to visualize these interactions in a near-native, vitrified state within cellular environments. However, the inherent radiation sensitivity of biological specimens imposes a fundamental constraint: the cumulative electron dose must be strictly managed to avoid beam-induced damage that destroys high-resolution information. This application note details practical strategies and protocols for low-dose imaging to maximize the fidelity of structural data, crucial for interpreting viral entry, replication, and egress mechanisms.

Quantitative Data on Beam-Induced Damage

Table 1: Critical Dose Limits for Biological Components in Cryo-ET

Biological Component Approx. Critical Dose (e⁻/Ų) Primary Damage Manifestation
Lipid Membranes ~50-100 Bubbling, loss of continuity
Protein Complexes (Secondary Structure) ~20-50 Loss of high-resolution features (>7Å)
Nucleic Acids (dsRNA/DNA) ~15-30 Strand breakage, density fragmentation
Cellular Context (Cytosol) ~10-20 Overall mass loss, contrast reduction
Viral Glycoproteins (Surface) ~15-40 Loss of peripheral density, blurring

Table 2: Comparative Performance of Low-Dose Strategies

Strategy Typical Total Dose (e⁻/Ų) Relative Information Retention Key Trade-off
Conventional Tomography 80-150 Low (High Damage) High SNR but severe structural alteration
Dose-Symmetric Tilt Scheme 40-80 Medium-High Improved high-resolution features in early tilts
Cryo-Conscious Autofocus 30-60 High Reduced overhead dose (~5-10 e⁻/Ų saved)
Volta Phase Plate Imaging 20-50 High at Low Dose Enhanced contrast, but plate alignment critical
Direct Electron Detector (DED) + Frame Alignment 20-60 Very High Enables dose fractionation, superior SNR/dose

Experimental Protocols

Protocol 1: Pre-Screening and Targeting Using Low-Magnification Atlas

Objective: Identify areas of interest (e.g., virus-bound cells) with minimal pre-exposure. Materials: Cryo-TEM with low-dose system, 200-300 mesh cryo-EM grid.

  • Load the cryo-grid into the holder under liquid nitrogen and insert into the microscope.
  • Set microscope to LowMag mode (e.g., 800x - 1500x). Use a defocused beam or beam-blanked search mode.
  • Navigate to a square of interest. Acquire a low-magnification (500x) overview map at <0.1 e⁻/Ų.
  • Switch to Mapping mode (~5,000x). Acquire a tile montage of the square using a dose of <1 e⁻/Ų total. Stitch images automatically.
  • Identify and record stage coordinates of target cells/virions using the software’s targeting function. Total pre-screening dose per target: <2 e⁻/Ų.

Protocol 2: Dose-Fractionated Cryo-Tomography Acquisition

Objective: Acquire a tilt series with optimal dose distribution for later sub-tomogram averaging. Materials: TEM with DED, automated tomography software (e.g., SerialEM, Tomo5).

  • Navigate to the target using beam-blanked stage movement.
  • Set Acquisition Parameters:
    • Acceleration Voltage: 300 kV.
    • Defocus: -6 to -8 µm (for phase contrast).
    • Pixel Size: 2.5-4.0 Å (calibrated).
    • Total Dose Budget: 40-60 e⁻/Ų.
    • Tilt Range: ±60° with 2° or 3° increment.
    • Dose-Symmetric Tilt Scheme: Acquire tilts as 0°, +1°, -1°, +2°, -2°, etc.
  • Calculate dose per frame/tilt: Divide total dose by number of tilts (e.g., 60 e⁻/Ų / 61 tilts ≈ 1 e⁻/Ų per tilt).
  • Set the DED to movie mode (e.g., 5-10 frames per tilt image). Adjust exposure time accordingly.
  • Initiate automated acquisition. The software will track features and adjust beam tilt.
  • Save all frame stacks (*.mrc) for each tilt for later motion correction.

Protocol 3: Cumulative Dose Tracking and Multi-Area Strategy

Objective: Ensure no area exceeds the critical dose, enabling multiple datasets per grid.

  • Enable the dose monitoring module in the acquisition software.
  • Define a grid map with multiple, non-overlapping areas of interest (AOIs).
  • After acquiring each tilt series at an AOI, the software logs the cumulative dose for that coordinate.
  • Program the software to skip any AOI that has already received a dose >10 e⁻/Ų from pre-screening.
  • Proceed to the next AOI. This systematic approach maximizes data yield per grid session.

Visualizations

Diagram 1: Low-Dose Cryo-ET Workflow for Host-Virus Samples

G START Vitrified Cell Sample on Cryo-EM Grid A1 Low-Mag Atlas (<0.1 e⁻/Ų) START->A1 A2 Mid-Mag Montage (<1 e⁻/Ų) A1->A2 A3 Target Selection (Virus-Cell Interface) A2->A3 B1 Beam-Blanked Move to Target A3->B1 B2 Dose-Symmetric Tilt Series Acquisition B1->B2 B3 Cumulative Dose Logged per Area B2->B3 C1 Frame Alignment & Motion Correction B3->C1 Dose-Fractionated Data C2 Tomogram Reconstruction C1->C2 C3 Subtomogram Averaging C2->C3 END 3D Molecular Architecture C3->END

Diagram 2: Cumulative Dose Management Logic

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Low-Dose Cryo-ET

Item Example Product/Type Critical Function in Low-Dose Imaging
Gold Fiducial Beads 10nm Protein A Gold, BSA Gold Tracer Provide high-contrast markers for tilt-series alignment with minimal added dose.
Quantifoil R2/2 Holey Carbon Grids Au 200 mesh, QF-R2/2 Standard grid for cellular cryo-ET. Consistent ice thickness aids targeting and reduces required search dose.
Ultra-Stable Cryo-TEM Holder Zeiss CRYO, Gatan 630 Minimizes drift during long acquisitions, preventing blurring and allowing lower dose rates.
Direct Electron Detector (DED) Gatan K3, Falcon 4, Selectris X Enables dose fractionation (movie mode); superior detective quantum efficiency (DQE) at low dose.
Volta Phase Plate Thermo Fisher Scientific VPP Increases contrast at low defocus, allowing acquisition at lower doses while maintaining interpretability.
Cryo-Plasma Cleaner Gatan Solarus, Quorum Gluve Hydrophilizes grid surface pre-blaming, ensuring even, thin ice to reduce required exposure.
Anti-Contaminator (Cold Finger) Integrated microscope system Maintains a cold surface near the sample to trap hydrocarbons, preventing contamination buildup during low-dose, long-duration imaging.
Automated Acquisition Software SerialEM, Tomo5, EPU Precisely controls beam blanking, stage movement, and dose distribution, enforcing dose budgets automatically.

Cryo-electron tomography (Cryo-ET) is an indispensable technique for visualizing the structural dynamics of host-virus interactions in a near-native state. A central challenge in applying Cryo-ET to thick cellular samples, such as virus-infected eukaryotic cells, is the inherent low contrast caused by weak phase shifts in the biological material and the inelastic scattering that contributes to a hazy background. This low signal-to-noise ratio obscures critical details of viral entry, replication, and assembly. This Application Note details two advanced electron microscopy technologies—Phase Plates and Energy Filtering—that dramatically improve contrast in thick cellular samples, thereby enabling high-fidelity 3D reconstruction of macromolecular complexes during infection. These protocols are framed within a doctoral thesis focused on elucidating the structural basis of herpesvirus capsid nuclear egress in primary fibroblasts.

Core Technologies: Principles and Quantitative Comparison

Volta Phase Plate (VPP) Technology

The Volta Phase Plate is a thin film of carbon that introduces a phase shift to the scattered electrons relative to the unscattered beam. A controlled electrostatic potential builds up on the plate when exposed to the electron beam, creating a phase shift near the back focal plane. This converts phase information into amplitude contrast, boosting the signal for low-spatial-frequency features critical for visualizing membranes and large complexes in crowded cellular environments.

Energy Filtering (Zero-Loss Filtering)

Energy filtering removes inelastically scattered electrons, which have lost energy through interactions with the sample and contribute to chromatic blur and a diffuse background. Using an in-column or post-column filter, only electrons that have lost negligible energy ("zero-loss" electrons) are used to form the image. This is particularly beneficial for samples thicker than ~300 nm, where inelastic scattering becomes pronounced.

Table 1: Quantitative Comparison of Contrast Enhancement Techniques for Cryo-ET

Parameter Volta Phase Plate (VPP) Energy Filtering (Zero-Loss) Conventional Defocus (Baseline)
Primary Mechanism Phase shift to amplitude contrast conversion Removal of inelastically scattered electrons Controlled underfocus (phase contrast)
Optimal Sample Thickness 200 - 500 nm >300 nm <200 nm
Typical Contrast Gain 2- to 5-fold increase in SNR at low spatial frequencies Up to 3-fold increase in SNR, reduces background by ~40% Baseline (1x)
Spatial Frequency Boost Maximal at low frequencies (<1/10 nm⁻¹) Uniform across frequencies High at mid-frequencies, oscillatory
Key Artifact/Consideration Phase plate charging, need for beam-induced potential stabilization Reduced signal intensity, requires higher dose or longer exposure Contrast transfer function (CTF) oscillations, requires correction
Impact on Dose Efficiency Higher contrast per electron, allows lower total dose Cleaner signal per electron, but total filtered signal is lower Lower efficiency, requires higher dose for same SNR

Experimental Protocols

Protocol 3.1: Integrated VPP and Energy Filtering for Tomography of Infected Cells

This protocol outlines the workflow for acquiring a high-contrast tomogram of a herpesvirus-infected cell using a Krios G4 microscope with a GIF and VPP.

I. Sample Preparation & Loading

  • Cell Culture & Infection: Seed primary human fibroblasts on glow-discharged, gold EM grids. Infect with HSV-1 at an MOI of 5 for 18 hours.
  • Cryo-Fixation: Vitrify grids using a plunge freezer (e.g., Leica GP2). Blot for 4-5 seconds at 95% humidity, 22°C, and plunge into liquid ethane.
  • Screening: Pre-screen grids on a 200kV screening microscope to locate regions of interest (cell periphery, ~400 nm thick) with viral particles.

II. Microscope Setup (Titan Krios G4 with GIF Quantum & VPP)

  • VPP Conditioning: Insert the VPP. Condition the phase plate by illuminating the central area with a 5 µm spot size at 300 kV for ~30 minutes until the phase shift stabilizes at π/2 (monitored by shift in Thon rings).
  • Energy Filter Alignment: Align the GIF. Set the slit width to 20 eV. Tune the zero-loss peak (ZLP) to the center of the slit using the spectrometer.

III. Tomography Data Acquisition

  • Low-Dose Navigation: Use the low-dose mode to navigate to a pre-identified target area.
  • Focus & Tuning: At a nearby area at the same height:
    • With VPP: Focus the image (not the back focal plane). Fine-tune the VPP bias to achieve optimal contrast.
    • With GIF: Acquire a spectrum, center the ZLP, and lock the slit.
  • Acquisition Scheme:
    • Tilt Series: -60° to +60° with 2° increment using a bidirectional scheme.
    • Defocus: -0.5 µm to -1.5 µm (additional to VPP for CTF stability).
    • Dose: Total dose 80-100 e⁻/Ų distributed per tilt using dose-symmetric tilt scheme.
    • Pixel Size: 3.4 Å.
    • Acquisition Time: ~30-40 minutes per tomogram.
  • Dual-Acquisition Strategy: For critical targets, acquire two sequential tilt series: one with VPP only and one with VPP+GIF for direct comparison.

Protocol 3.2: Contrast Transfer Function (CTF) Determination for VPP Data

CTF estimation is crucial for subsequent reconstruction and subtomogram averaging.

  • Tilt Series Alignment: Align the tilt series using fiducial-less or patch-tracking algorithms (e.g., in IMOD).
  • CTF Estimation per Tilt: Use a dedicated algorithm for phase plate data (e.g., ctfplotter in EMAN2 or ctffind_pp). Key parameter: set the expected phase shift to 90° (π/2).
  • CTF Correction: Apply phase-flipping or Wiener filtering during 3D reconstruction using the estimated CTF parameters.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for High-Contrast Cryo-ET of Host-Virus Samples

Item Function/Application
Quantifoil R2/2, 300 mesh, Au grids Gold grids provide better conductivity, reducing charging. R2/2 hole size is ideal for cellular samples.
Liquid Ethane (99.999% purity) Primary cryogen for rapid vitrification, preventing crystalline ice formation.
FIB-SEM System (e.g., Thermo Scientific Aquilos 2) For preparing lamellae (~200-300 nm thick) from specific cellular regions via cryo-FIB milling.
Holey Carbon Film (for VPP tuning) Used for acquiring Thon rings to monitor and adjust the VPP phase shift during conditioning.
Fiducial Gold Beads (10-15 nm) Optional for tilt series alignment, though less critical with modern patch-tracking.
Cryo-ET Processing Software Suite (IMOD, TomoBEAR, M) For alignment, reconstruction, CTF correction, denoising, and subtomogram averaging.
Direct Electron Detector (e.g., Falcon 4) High detective quantum efficiency (DQE) camera essential for low-dose, high-fidelity acquisition.

Diagrams

workflow START Sample: HSV-1 Infected Cell on Grid A Vitrification (Plunge Freezing) START->A B Cryo-FIB Milling (Optional for thick cells) A->B C Screening (200kV TEM) B->C D High-Resolution Tomo. Setup C->D PP VPP Conditioning & Tuning D->PP EF Energy Filter Alignment (20eV slit) D->EF E Acquire Tilt Series (-60° to +60°, 2°) PP->E EF->E F CTF Estimation & Correction E->F G Tomogram Reconstruction F->G H Segmentation & Analysis G->H

Title: Cryo-ET Workflow with VPP & Energy Filtering

signaling Virus Virus Attachment Attachment Virus->Attachment MembFusion MembFusion Attachment->MembFusion CapsidTransport CapsidTransport MembFusion->CapsidTransport HostMembrane Host Cell Membrane MembFusion->HostMembrane Binds NuclearPore NuclearPore CapsidTransport->NuclearPore Cytoskeleton Cytoskeleton CapsidTransport->Cytoskeleton Utilizes Replication Replication NuclearPore->Replication NPC Nuclear Pore Complex (NPC) NuclearPore->NPC Docks at CapsidAssembly CapsidAssembly Replication->CapsidAssembly Envelopment Envelopment CapsidAssembly->Envelopment Egress Egress Envelopment->Egress NEC Nuclear Egress Complex (NEC) Envelopment->NEC Budding via

Title: Herpesvirus Lifecycle & Host Structures Imaged by Cryo-ET

tech_compare Problem Low Contrast in Thick Cellular Cryo-ET Solution1 Phase Plate (VPP) Problem->Solution1 Solution2 Energy Filtering Problem->Solution2 Mech1 Mechanism: Phase Shift → Amplitude Contrast Solution1->Mech1 Benefit1 Benefit: Boosts Low-Frequency Detail (Membranes, Large Complexes) Mech1->Benefit1 Outcome Synergistic Outcome: High-Fidelity 3D Reconstruction of Host-Virus Interfaces Benefit1->Outcome Mech2 Mechanism: Removes Inelastic Scattering Solution2->Mech2 Benefit2 Benefit: Reduces Background Haze Improves SNR in Thick Samples Mech2->Benefit2 Benefit2->Outcome

Title: Contrast Enhancement Technology Logic

Within the broader thesis on Cryo-Electron Tomography (cryo-ET) for imaging host-virus interactions, the missing wedge problem represents a fundamental technical limitation. Cryo-ET involves acquiring a series of 2D projection images of a vitrified biological sample, such as a virus-infected cell, tilted along a single axis. Due to physical constraints of the microscope stage and sample geometry, the tilt series is typically limited to a range of approximately ±60°, leaving a missing wedge of information in Fourier space. This anisotropic lack of data leads to distortions and reduced resolution in the final 3D reconstruction, directly impacting the interpretability of macromolecular structures, such as viral fusion machinery or host immune complexes.

Quantitative Impact on Resolution and Fidelity

The following table summarizes the empirically characterized impacts of the missing wedge on tomographic reconstructions, crucial for quantifying limitations in host-virus interaction studies.

Table 1: Quantitative Effects of the Missing Wedge on Tomographic Reconstructions

Parameter Full Tilt Range (±90°) Typical Tilt Range (±60°) Impact on Host-Virus Imaging
Isotropic Resolution Achievable Reduced by ~30% along Z-axis Compromises visualization of viral spike protein dimensions.
Anisotropy Factor 1.0 (Isotropic) ~1.5-2.0 (Anisotropic) Elongation artifacts perpendicular to tilt axis distort membrane structures.
Fourier Space Coverage 100% ~66% Missing information hinders high-fidelity classification of protein conformations.
Signal-to-Noise Ratio (SNR) Optimal Degraded due to incomplete projection Challenges detection of small, flexible host factors bound to virions.
Reconstruction Fidelity (FSC₀.₅) High Can decrease by 15-25% Limits resolvability of key interfaces for drug target identification.

Protocols for Mitigation and Correction

Protocol 3.1: Dual-Axis Tomography for Improved Coverage

Objective: To reduce the missing wedge to a missing pyramid by acquiring a second, orthogonal tilt series, thereby improving isotropy.

Materials:

  • Cryo-electron microscope equipped with a high-tilt, auto-loading holder.
  • Fiducial gold markers (e.g., 10nm colloidal gold).
  • Automated acquisition software (e.g., SerialEM, Tomography 5).

Procedure:

  • Grid Preparation: Apply a high density of fiducial gold markers to the cryo-grid prior to vitrification.
  • First Axis Acquisition: Acquire a tilt series from -60° to +60° with a 2-3° increment.
  • Grid Rotation: In the microscope, rotate the grid precisely by 90°.
  • Second Axis Acquisition: Acquire a second, orthogonal tilt series over the same angular range.
  • Fiducial Alignment: Use fiducial markers to align projection images from both series independently.
  • Reconstruction and Merge: Reconstruct two tomograms (e.g., using IMOD). Combine the volumes by merging in Fourier space or using iterative real-space algorithms to generate a dual-axis tomogram.

Protocol 3.2: Sub-tomogram Averaging (STA) and Classification

Objective: To achieve high-resolution structures by aligning and averaging thousands of extracted, identical particles, mitigating missing wedge effects through averaging.

Materials:

  • Tomogram reconstruction software (IMOD, AreTomo).
  • Sub-tomogram averaging suite (M, RELION, emClarity).
  • High-performance computing cluster.

Procedure:

  • Initial Tomogram Reconstruction: Generate tomograms using weighted back-projection or SIRT-like algorithms.
  • Particle Picking: Manually or semi-automatically pick putative viral or host complex particles from multiple tomograms.
  • Initial Alignment: Perform iterative 3D alignment and averaging to generate an initial, low-resolution reference model, compensating for missing wedge effects during alignment.
  • 3D Classification: Use masked classifications to separate heterogeneous states (e.g., pre-fusion vs. post-fusion viral glycoproteins).
  • High-Resolution Refinement: Refine aligned particles against the generated references, applying missing wedge correction in the refinement steps, to produce a final, higher-fidelity map.

Protocol 3.3: Missing Wedge Compensation via Iterative Reconstruction

Objective: To improve reconstruction fidelity using iterative algorithms that incorporate constraints.

Materials:

  • Software for iterative reconstruction (TOM Toolbox, Protomo, SIRT implementation in IMOD).
  • High-performance GPU workstations for computation.

Procedure:

  • Generate Initial Reconstruction: Create a first-pass reconstruction using weighted back-projection.
  • Apply Real-Space Constraints: Impose constraints such as positivity (density ≥ 0) and a defined mask/boundary (e.g., the known shape of a virion).
  • Forward Projection: Re-project the constrained 3D volume to simulate 2D projections at the original tilt angles.
  • Update Projections: Replace the simulated projections' corresponding missing wedge regions with the original experimental data.
  • Back-Projection & Iteration: Reconstruct a new 3D volume from the updated projections. Repeat steps 2-5 for 20-50 iterations until convergence.

Diagrams

Diagram 1: Missing Wedge in Fourier Space

G P1 Tilt Series (±60°) F1 2D Fourier Transforms P1->F1 Transform FW Fourier Space F1->FW Map MW Missing Wedge FW->MW Missing Data T1 Anisotropic 3D Tomogram FW->T1 Inverse Transform

Diagram 2: Dual-Axis Tomography Workflow

G Start Cryo-Sample with Gold Fiducials TS1 Axis 1 Tilt Series -60° to +60° Start->TS1 Rotate In-Situ Grid Rotation (90°) TS1->Rotate TS2 Axis 2 Tilt Series -60° to +60° Rotate->TS2 Align Dual-Axis Alignment & Merge TS2->Align Tomo Isotropically Improved Tomogram Align->Tomo

Diagram 3: Sub-tomogram Averaging Pipeline

G T Input Tomograms (with Missing Wedge) P Particle Picking T->P IA Initial Alignment & Averaging P->IA C 3D Classification & Heterogeneity Analysis IA->C R High-Resolution Refinement C->R O Final Averaged Map (Enhanced Fidelity) R->O

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Cryo-ET Studies of Host-Virus Interactions

Item Function/Application Example Product/Type
Quantifoil or UltraAufoil Grids Holey carbon film grids provide thin, stable vitreous ice for cellular tomography. Quantifoil R2/2, 300 mesh, Au.
Colloidal Gold Fiducials High-contrast markers for precise alignment of tilt series images. 10-15nm Protein A-gold or plain colloidal gold.
Vitrification Robot Ensures rapid, reproducible, and consistent plunge-freezing to create amorphous ice. Thermo Fisher Vitrobot Mark IV, Leica GP2.
Phase Plate Enhances contrast of biological specimens, critical for low-dose imaging of cellular landscapes. Volta Phase Plate, Zernike phase plate.
Cryo-FIB/SEM System Prepares thin lamellae (~200nm) from vitrified cells for in-situ tomography of infection sites. Thermo Fisher Scios 2 Cryo-FIB, Teneo Volumescope.
Acquisition Software Automated software for collecting low-dose, multi-scale tilt series. SerialEM, Tomography 5 (Thermo Fisher).
Processing Suite Comprehensive software package for tilt-series alignment, reconstruction, and analysis. IMOD, including eTomo, 3dmod.
Sub-tomogram Averaging Package Software for extracting, aligning, averaging, and classifying macromolecular complexes. RELION, M, emClarity, Dynamo.

1. Introduction & Thesis Context Within a broader thesis on Cryo-Electron Tomography (Cryo-ET) for imaging host-virus interactions, the preparation of artifact-free, electron-transparent lamellae is the critical bottleneck. Focused Ion Beam (FIB) milling at cryogenic temperatures must be optimized to preserve ultrastructural details of viral entry, replication, and egress. This protocol details the systematic optimization of FIB parameters to achieve consistency and high yield in lamella production, directly enabling high-resolution structural studies of viral infection mechanisms and supporting targeted therapeutic development.

2. Key FIB-Milling Parameters: Quantitative Summary The primary adjustable parameters in cryo-FIB milling, their typical ranges, and their impact on lamella quality are summarized below.

Table 1: Core FIB-Milling Parameters and Optimization Targets

Parameter Typical Range Effect on Lamella Quality Optimization Goal
Ion Beam Current 1 pA - 1 nA Lower current (<50 pA) for fine polishing; higher current for bulk material removal. Minimize curtaining, reduce amorphization.
Accelerating Voltage 30 kV (standard) Lower voltage (e.g., 5-8 kV) reduces Ga+ implantation and damage. Reduce subsurface damage layer.
Milling Pattern & Angle Pre-tilt: 8°-12°; Cleaning cross-section: ±3°-5° Symmetrical cleaning patterns reduce wedge angle and taper. Achieve parallel, uniform thickness.
Lamella Target Thickness 150 - 300 nm Thinner lamellae (150-200 nm) yield better tomogram resolution but are fragile. Balance between electron transparency and mechanical stability.
Final Polish Current < 10 pA (often 1-5 pA) Defines the final surface quality. Critical for removing redeposited material. Produce smooth, curtaining-free surfaces.
Gas Injection System (GIS) Pt, Organometallic precursors Deposition of protective layer prior to milling. Uniformity is key. Ensure homogeneous protection of region of interest.

Table 2: Troubleshooting Common Lamella Artifacts

Artifact Likely Cause Corrective Parameter Adjustment
Curtaining (Vertical Streaks) Uneven initial surface or high beam current during bulk milling. Apply a thicker, uniform protective layer; use lower current for bulk milling; implement multi-step milling.
Wedge Shape / Tapering Asymmetric milling angles or uneven material. Use symmetrical cleaning cross-section patterns; ensure stage eucentricity is perfectly calibrated.
Amorphous Surface Layer Excessive ion beam dose, high final current. Implement a low-kV (5-8 kV) final polish step; reduce final polish beam current.
Lamella Fracture Too thin target thickness; thermal or mechanical stress. Increase target thickness to 200-250 nm; ensure stable cryo-conditions; refine milling strategy.

3. Detailed Experimental Protocol: Sequential FIB-Milling for Host-Cell Lamellae

Protocol: Cryo-FIB Lamellization of Virus-Infected Cells Objective: To produce a 200 nm thick, parallel-sided lamella from a vitrified cell monolayer containing a region of host-virus interaction.

A. Pre-Milling Preparation

  • Sample: Vitrified RBD (Rodel Buffer Dish) with virus-infected cell monolayer on EM grid.
  • Loading: Transfer grid under liquid nitrogen to cryo-FIB/SEM microscope shuttle. Insert into pre-cooled (< -170°C) stage.
  • Localization: Using SEM at 2-5 kV, low current, navigate to a cell of interest. Use fiducial markers or morphological cues to identify potential infection sites (e.g., syncytia, vesicle clusters).
  • Protective Coating:
    • Move stage to 0° tilt. Using the Gas Injection System (GIS), pre-clean the deposition needle.
    • Navigate to region of interest (ROI). Tilt stage to 8-12° (typical).
    • Deposit a uniform, rectangular organometallic Pt layer (~1-2 µm thick) over the ROI using the ion beam (e.g., 30 kV, 0.3 nA). Ensure coverage extends beyond the planned lamella boundaries.

B. Bulk Milling (Rough Lamella Definition)

  • Stage Tilt: Position stage so the ion beam is perpendicular to the sample surface for the trench milling step (often ~8° relative to the stage normal).
  • Front Trench: Define a rectangular trench in front of the Pt-protected ROI. Use a relatively high current (e.g., 0.5-1 nA at 30 kV) to rapidly remove material. Stop ~5 µm from the lamella plane.
  • Back Trench & Undercut: Define a deeper trench behind the lamella. Perform a "U-cut" or "Lift-out cut" beneath the lamella to isolate it, leaving attachment points at the top and bottom.

C. Fine Milling & Thinning

  • Stepwise Thinning: Thin the lamella using sequentially lower ion beam currents (e.g., 100 pA -> 50 pA -> 30 pA). Mill from both sides using symmetrical cleaning cross-section patterns (e.g., ±3°).
  • Thickness Monitoring: Use the SEM to periodically image the lamella edge at high contrast. Estimate thickness by measuring the shadow cast or using built-in measurement tools. Target ~500 nm at this stage.
  • Final Polish: Perform the final thinning to target thickness (200 nm) using a very low beam current (<10 pA, ideally 1-5 pA). Consider switching to a lower accelerating voltage (5-8 kV) for this step to minimize ion damage.

D. Post-Milling Verification

  • Image the lamella at high resolution in SEM mode (3-5 kV) to check for smoothness, absence of curtains, and uniform thickness.
  • Record all milling parameters (currents, angles, times) for the lamella in a lab notebook or database for correlation with subsequent tomogram quality.

4. The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key Materials for Cryo-FIB Lamella Preparation

Item / Reagent Function in Experiment
Quantifoil or C-flat EM Grids (Au, 200 mesh) Support film for cell culture growth and vitrification. Gold is non-magnetic and conductive.
Liquid Ethane / Propane Cryogen Used for rapid plunge vitrification to preserve cellular structures in a near-native, amorphous ice state.
Cryo-Plunge Freezer (e.g., Vitrobot, GP2) Instrument for controlled blotting and rapid immersion of the grid into cryogen for vitrification.
Organometallic Gas (e.g., Pt precursor) Injected to deposit a protective layer over the region of interest prior to ion beam milling, preventing surface damage.
Cryo-TEM Grid Storage Box For safe, organized, and contamination-free storage and transfer of vitrified grids under liquid nitrogen.
Anti-Contaminator (Cold Trap) A liquid nitrogen-cooled surface within the FIB/SEM vacuum chamber to trap hydrocarbons and prevent ice contamination on the sample.
Fiducial Gold Beads (e.g., 10-15 nm) Applied to grid surface prior to vitrification. Serve as reference markers for tomogram alignment and reconstruction during data processing.

5. Visualized Workflows & Relationships

G cluster_0 FIB-Milling Optimization Loop VirusInfection Virus-Infected Host Cells Vitrification Cryo-Plunge Vitrification VirusInfection->Vitrification FIBPrep Cryo-FIB/SEM Transfer Vitrification->FIBPrep GISDeposit GIS Protective Coating FIBPrep->GISDeposit BulkMill Bulk Milling (High Current) GISDeposit->BulkMill ThinPolish Thinning & Polish (Low kV/pA) BulkMill->ThinPolish Lamella 200nm Lamella ThinPolish->Lamella CryoET Cryo-ET Data Acquisition Lamella->CryoET ThesisGoal 3D Host-Virus Interaction Models CryoET->ThesisGoal

Diagram 1: Lamella Prep Workflow for Host-Virus Cryo-ET

G Param Key Parameter Adjustment Current Beam Current ↓ (High -> Low) Param->Current Voltage Accel. Voltage ↓ (30kV -> 5kV) Param->Voltage Symmetry Milling Symmetry ↑ Param->Symmetry Artifact1 Reduced Curtaining Current->Artifact1 Artifact2 Reduced Amorphous Layer Voltage->Artifact2 Artifact3 Reduced Tapering Symmetry->Artifact3 Outcome High-Quality Lamella Artifact1->Outcome Artifact2->Outcome Artifact3->Outcome

Diagram 2: Parameter Effects on Lamella Artifacts

Application Notes: Current Landscape and Quantitative Benchmarks

The computational demands of cryo-electron tomography (cryo-ET) for host-virus research are defined by data volume, velocity, and processing complexity. The following tables summarize current quantitative benchmarks.

Table 1: Cryo-ET Data Generation Metrics for Host-Virus Studies

Data Generation Stage Typical Data Volume per Tomogram Current High-End Benchmark Primary Computational Load
Raw Tilt-Series Acquisition 40-80 GB (70+ images, 4k x 4k, 16-bit) 150+ GB (Dose-fractionated, 8k x 8k) Storage I/O, Real-time Pre-processing
Tomogram Reconstruction (Weighted Back Projection) 10-15 GB (4k x 4k x 500 voxels, 32-bit float) 60+ GB (8k x 8k x 1000) GPU Memory, High-bandwidth RAM
Subtomogram Averaging (Class of 10k particles) 2-5 TB (intermediate alignment files) 20+ TB (Large-scale asymmetric refinements) Parallel File System, Multi-node CPU/GPU
Final Atomic Model & Map 1-5 GB 10+ GB Visualization RAM, Specialist GPU

Table 2: Comparison of Processing Pipeline Architectures (2024)

Pipeline Architecture Pros Cons Best Suited For
Monolithic (e.g., IMOD, Tomo3D) Integrated, standardized workflows. Lower initial setup complexity. Scaling bottlenecks. Hard to customize for novel algorithms. Single-workstation processing, standard reconstructions.
Modular Script-based (e.g., RELION, M, Warp) Flexibility, mix-and-match best algorithms. Community-driven updates. Requires extensive scripting/user expertise. Data transfer overhead between modules. Research groups with computational support, novel method development.
Cloud-Native (e.g., CryoCloud, Leginon/TEMography on AWS) Elastic scaling, no local hardware burden. Enhanced collaboration. Ongoing cost, data egress fees. Potential latency for interactive steps. Multi-institutional projects, burst processing, lacking local HPC.
Hybrid High-Performance Computing (HPC) Maximum processing power for subtomogram averaging. Handles largest datasets. Queue times, requires parallel programming expertise (MPI). Final high-resolution refinements of massive particle sets.

Detailed Protocols

Protocol 2.1: Streamlined Cryo-ET Processing Pipeline for High-Throughput Host-Virus Samples

Objective: To reconstruct and denoise tomograms from tilt-series data for initial visualization of virus entry or budding events, optimized for throughput.

Materials & Software:

  • Raw dose-fractionated tilt-series (.mrc or .tiff).
  • Workstation with ≥ 2 high-end GPUs (e.g., NVIDIA A100/A6000), 512GB RAM, fast NVMe storage.
  • Software: SerialEM (acquisition), Warp, IMOD, M, Topaz.

Procedure:

  • Pre-processing & Motion Correction (Warp):
    • Import raw movie stacks into Warp.
    • Execute warp_preprocess script with dose-weighting enabled (total dose ~80-100 e⁻/Ų). Align frames using patch-based motion correction.
    • Output: Motion-corrected, dose-weighted micrographs for each tilt angle.

  • Tilt-Series Alignment and CTF Estimation (M or IMOD):

    • In M, use the motioncor2 and ctffind wrappers per tilt. Generate a preliminary alignment model using patch-tracking.
    • Alternatively, in IMOD, run alignframes and ctfplotter. Use patchtrack for coarse alignment, then autofidseed and fidtrack for fine alignment with fiducial gold beads.
    • Output: .tlt or .rawtlt file (tilt angles), .xf file (transformations).

  • Tomogram Reconstruction (IMOD or TOOL):

    • In IMOD, execute: tilt -InputProjections aligned_stack.mrc -OutputFile reconstruction.mrc -TILTFILE angles.tlt -THICKNESS 1200 -RotationAxis 4.5 -FULLIMAGE 4096 4096.
    • Use weighted back-projection (WBP) for speed or SIRT-like (e.g., SART) for initial quality.
    • Output: 3D reconstruction file (.mrc).

  • Tomogram Denoising (Topaz or IsoNet):

    • For Topaz: Run topaz denoise3d -m model_name -o denoised.mrc reconstruction.mrc. Use a model pre-trained on cryo-ET data.
    • For IsoNet: Run iterative missing wedge correction and denoising cycles using the IsoNet CNN architecture.
    • Output: Denoised, interpretable tomogram ready for segmentation.

Protocol 2.2: Distributed Subtomogram Averaging Pipeline on an HPC Cluster

Objective: To average thousands of extracted viral glycoprotein complexes from infected cell tomograms to achieve sub-nanometer resolution.

Materials & Software:

  • Directory of reconstructed, denoised tomograms (.mrc).
  • Initial particle coordinates (.box or .coord files).
  • HPC cluster with SLURM scheduler, high-speed parallel filesystem (e.g., Lustre, BeeGFS), multiple GPU nodes.
  • Software: RELION, Dynamo, MPI libraries.

Procedure:

  • Particle Extraction & Initialization (Dynamo):
    • Create a dynamo catalog. Use dynamo_table_extract to cut out subvolumes based on coordinate tables.
    • Generate an initial reference, either from previous work or by low-pass filtering a crude average.
    • Output: Particle stack (.mrcs), initial reference (.mrc), star file (.star).

  • Job Preparation for RELION on HPC:

    • Prepare a relion_refine command template. Critical parameters: --iter 25 --tau2_fudge 2 --oversampling 1 --healpix_order 2 --offset_range 5 --offset_step 2 --sym C1 --norm --scale --ctf.
    • Create a SLURM submission script that requests multiple nodes and GPUs, using mpirun -n 160 relion_refine_mpi ....

  • Iterative Alignment & Classification (RELION):

    • Submit the 3D auto-refinement job. RELION will perform expectation-maximization, distributing Fourier-space operations across MPI processes.
    • Monitor run_model.star and run_data.star for resolution progress (FSC 0.143 criterion).
    • After refinement, perform 3D classification (relion_refine --class3d) without alignment to isolate heterogeneous states (e.g., pre-fusion vs. post-fusion viral spikes).

  • Post-processing & Analysis:

    • Run relion_postprocess to sharpen the final map, apply a mask, and calculate the final resolution.
    • Use ucsf_chimera or cryoSPARC for visualization and model docking.

Visualizations

G start Raw Tilt-Series (70+ images, ~80 GB) proc1 Motion Correction & Dose Weighting (Warp) start->proc1 proc2 Tilt-Series Alignment & CTF Estimation (M/IMOD) proc1->proc2 proc3 Tomogram Reconstruction (WBP/SIRT) proc2->proc3 proc4 Denoising & Missing Wedge Correction (Topaz/IsoNet) proc3->proc4 branch Analysis Branch proc4->branch seg Segmentation & Feature Detection (EMAN2, Chimera) branch->seg  Single Tomogram avg Subtomogram Averaging Pipeline (RELION/Dynamo) branch->avg  Particle Set vis Visualization & Interpretation (Chimera, UCSF) seg->vis avg->vis end Atomic Model & Biological Insights vis->end

Title: Cryo-ET Data Processing and Analysis Workflow

G cluster_compute Compute Farm user Researcher Workstation transfer High-Speed Data Transfer Node user->transfer Globus/rsync Raw Data head Cluster Head Node (Job Scheduler: SLURM) user->head SSH Job Submission storage Parallel Filesystem (Lustre/BeeGFS) ~2-10 PB, High I/O transfer->storage Ingest head->storage gpu1 GPU Node 1 (4x A100, 512GB RAM) head->gpu1 MPI Job Launch gpu2 GPU Node 2 (4x A100, 512GB RAM) head->gpu2 cpu1 High-Memory CPU Node (2TB RAM) head->cpu1 gpu1->storage I/O gpu2->storage I/O cpu1->storage I/O

Title: Hybrid HPC-Cluster Architecture for Cryo-ET

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Computational Tools for Cryo-ET Host-Virus Research

Item/Software Function/Application Key Consideration for Pipeline Integration
Warp Automated preprocessing: motion correction, dose-weighting, CTF estimation, particle picking. Dramatically reduces manual time; outputs compatible with RELION/M. Essential for high-throughput.
RELION Bayesian subtomogram averaging, classification, and refinement. Industry standard for high-resolution. Requires HPC/GPU cluster for non-trivial datasets. MPI implementation is efficient but needs expert setup.
IMOD Comprehensive suite for tilt-series alignment, tomogram reconstruction, and basic segmentation. Robust, well-documented. etomo GUI is user-friendly but command-line is needed for batch processing.
UCSF Chimera/X Visualization, segmentation, fitting of atomic models into tomographic maps. Crucial for interpretation. Scriptable (chimera.cmd) for batch operations like map segmentation.
Topaz Deep learning tool for particle picking and tomogram denoising. Reduces manual picking from days to hours. Requires training or pre-trained models on appropriate data.
Dynamo Toolbox for subtomogram averaging, alignment, and classification. Flexible for complex geometries (e.g., membrane curvature). Steeper learning curve but powerful for in-situ work.
CryoSPARC SaaS and local software for processing, includes cryo-ET tools (Patch Tracking, STA). Rapid, user-friendly live processing. Licensing cost. Streamlined but less customizable than RELION.
Slurm/Altair PBS Pro Workload manager for HPC clusters. Essential for scheduling and resource allocation. Scripting knowledge required to optimize job arrays for thousands of tilt-series.
Parallel Filesystem (e.g., Lustre) High-speed, scalable storage for simultaneous read/write by hundreds of processes. Critical for STA performance. I/O bottlenecks can idle expensive GPU nodes. Must be tuned.
AWS/Azure/GCP Cloud Cloud computing platforms offering scalable, on-demand resources and specialized VM images. Eliminates capital hardware cost. Ideal for burst capacity or standardized, portable pipelines. Cost monitoring is essential.

Benchmarking Cryo-ET: How It Stacks Up Against X-ray Crystallography and Single-Particle Cryo-EM

Within the broader context of cryo-electron tomography (cryo-ET) for host-virus interaction research, selecting the optimal imaging technique is critical. Each method offers distinct trade-offs between spatial resolution, temporal resolution (throughput), and sample requirements. This application note provides a comparative analysis of key structural and cellular imaging modalities, including detailed protocols for their application in virology.

Quantitative Comparison of Major Techniques

The following table summarizes the core performance metrics of major imaging techniques relevant to structural virology and cell biology.

Table 1: Technique Comparison: Resolution, Throughput, and Sample Needs

Technique Typical Resolution (3D) Throughput (Data to Publishable Map) Sample Requirements & Key Constraints Primary Application in Host-Virus Research
X-ray Crystallography 1.5 – 3.0 Å Medium-High (if crystals are available) Requires high-purity, homogeneous protein that forms large, ordered crystals. Cannot image complexes in situ. Atomic structure of purified viral proteins, protein-drug complexes.
Single-Particle Cryo-EM (SPA) 2.0 – 4.0 Å (for >100 kDa) Medium (weeks to months) Requires high-purity, homogeneous protein in solution (≥ 50 kDa ideal). Sensitive to conformational heterogeneity. High-res structures of purified viral capsids, spikes, or soluble host receptors.
Cryo-Electron Tomography (cryo-ET) 15 – 40 Å (for subtomogram averaging) Low (months, high effort) Intact cells or vitrified lamellae (≤ 500 nm thick). Low signal-to-noise. Targets must be repetitive for averaging. Visualizing virus entry, assembly, and egress in situ within host cellular context.
Cryo-Focused Ion Beam Milling (Cryo-FIB) + cryo-ET 15 – 40 Å (after averaging) Very Low (highly specialized workflow) Requires vitrified cells on a grid. Milling produces ~200 nm lamellae. Technically demanding, low throughput. Creating thin lamellae from thick cells/organelles for in situ cryo-ET of infection sites.
Super-Resolution Light Microscopy (e.g., STED, PALM) 20 – 100 nm (lateral) High (live-cell imaging possible) Requires specific fluorescent labeling (antibodies, tags). Resolution limited by photon budget and label size. Live-cell tracking of viral particles, co-localization studies of host/viral components.

Experimental Protocols

Protocol 2.1: Single-Particle Analysis (SPA) of a Viral Glycoprotein

Objective: Determine the high-resolution structure of a purified recombinant viral spike protein.

  • Sample Preparation: Thaw purified glycoprotein (≥ 0.5 mg/mL in mild buffer). Apply 3 µL to a glow-discharged holey carbon grid (Quantifoil R1.2/1.3), blot for 3-5 seconds (100% humidity, 4°C), and plunge-freeze in liquid ethane using a vitrification device (e.g., Vitrobot).
  • Data Collection: Load grid into a 300 keV cryo-TEM (e.g., Titan Krios). Using a direct electron detector (e.g., Gatan K3) in counting mode, collect ~5,000 micrographs at 81,000x magnification (pixel size 1.05 Å). Use a defocus range of -0.8 to -2.5 µm. Total dose ≤ 50 e⁻/Ų.
  • Image Processing: Use RELION or cryoSPARC. Perform patch motion correction and CTF estimation. Autopick particles, extract, and perform 2D classification to remove junk. Several rounds of 3D classification and heterogeneous refinement are required to separate conformational states. Final homogeneous refinement with imposed symmetry (if applicable) and Bayesian polishing yields the final map.
  • Model Building: Dock an existing homology model into the map using Chimera. Manually rebuild and real-space refine in Coot, followed by iterative refinement in Phenix.

Protocol 2.2: Cryo-ET of Virus-Infected Cells via Cryo-FIB Milling

Objective: Visualize the ultrastructure of viral assembly factories inside infected cells.

  • Cell Vitrification: Seed cells on glow-discharged gold EM grids (e.g., R2/2). Infect at desired MOI. At the target infection time, plunge-freeze grid using a vitrification device.
  • Cryo-FIB Milling: Transfer grid under liquid nitrogen to a dual-beam FIB/SEM (e.g., Thermo Scientific Scios 2 Cryo-FIB). Sputter-coat with organometallic platinum for 15 seconds. Identify a target cell using the SEM at 5 keV, 13 pA. Mill trenches on either side of the cell using the Ga⁺ FIB at 30 keV, 1-50 pA, progressively lowering current. Polish the final lamella to ~200 nm thickness using a 30 pA then 10 pA beam.
  • Tomography Data Collection: Transfer lamella to a 300 keV cryo-TEM. Using a dose-symmetric tilt scheme (e.g., from -60° to +60° with 3° increment), collect a tilt series at 26,000x (pixel size 5.3 Å) with a total dose of 120-150 e⁻/Ų. Use a phase plate or Volta phase plate if available to enhance contrast.
  • Tomogram Reconstruction & Analysis: Align tilt series using patch tracking (e.g., in IMOD). Reconstruct tomogram via weighted back-projection or SIRT-like algorithms (in IMOD or AreTomo). Denoise using deep-learning tools (Topaz, IsoNet). Manually segment or use template matching to identify and average sub-tomograms of repeating viral structures.

G Start Virus-infected Cells on EM Grid Vitrification Plunge Freezing (Vitrification) Start->Vitrification CryoFIB Cryo-FIB Milling (Produce ~200 nm Lamella) Vitrification->CryoFIB TomoAcquire Cryo-ET Data Collection (Tilt Series Acquisition) CryoFIB->TomoAcquire Reconstruct Tomogram Reconstruction & Denoising TomoAcquire->Reconstruct Analyze Segmentation & Subtomogram Averaging Reconstruct->Analyze Result 3D Visualization of Host-Virus Interfaces Analyze->Result

Title: Workflow for In Situ Cryo-ET via Cryo-FIB Milling

Protocol 2.3: Correlative Light and Electron Microscopy (CLEM) for Targeting

Objective: Locate rare virus-infected cell events for targeted cryo-FIB milling.

  • Fluorescent Labeling: Infect cells expressing a fluorescently tagged host protein (e.g., GFP-Rab7) with viruses containing a differently tagged protein (e.g., mCherry-VP40). Culture on a finder grid.
  • Live-Cell Imaging: Image grid using a spinning-disk confocal or widefield fluorescence microscope equipped with an environmental chamber. Acquire Z-stacks to identify cells with the desired interaction (e.g., colocalization of GFP and mCherry signals).
  • Correlation: Record the grid coordinates (e.g., using a FinderGrid pattern) and precise XYZ stage positions of target cells.
  • Cryo-Fixation: Immediately plunge-freeze the grid. Remount and transfer to a cryo-light microscope (cryo-FLM) to re-identify the fluorescent target under cryogenic conditions and record updated coordinates.
  • Targeted Milling: Transfer grid to the cryo-FIB/SEM. Use the cryo-FLM map to navigate to the target cell with high precision before proceeding with milling (Protocol 2.2).

G Label Fluorescently Labeled Sample LiveLM Live-Cell Light Microscopy (Identify Event) Label->LiveLM Map Create Correlation Map & Coordinates LiveLM->Map Cryo Plunge Freeze Map->Cryo CryoLM Cryo-Light Microscopy (Confirm Target) Cryo->CryoLM FIB Cryo-FIB/SEM (Targeted Milling) CryoLM->FIB Tomo Cryo-ET FIB->Tomo

Title: Correlative Light and Electron Microscopy (CLEM) Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Cryo-ET of Host-Virus Interactions

Item Function & Rationale
Quantifoil or UltrAuFoil Gold Grids EM support grids. Gold is inert and conducts heat well. Holey carbon or ultraflat gold surfaces provide optimal ice thickness and stability.
Cryo-FIB Autogrids Specialized grids with a clipping ring compatible with specific FIB/SEM and TEM stages, ensuring secure lamella transfer.
Liquid Ethane Propane Mix Cryogen for plunge-freezing. Provides rapid cooling rates (>10⁴ K/sec) to achieve vitreous (non-crystalline) ice, preserving native structure.
Fiducial Gold Beads (e.g., 10 nm Protein A Gold) Added to sample prior to tilt-series collection. Serve as reference markers for accurate alignment of tilt series images during tomogram reconstruction.
Phase Plate (Volta or Zach) TEM accessory that shifts the phase of unscattered electrons, dramatically enhancing image contrast at low dose, critical for beam-sensitive biological samples.
Cryo-EM Density Modification Reagents (e.g., GraFix) Glycerol gradient fixation/stabilization. Can be used for some samples to improve particle homogeneity and stability before grid freezing, though not for true native-state studies.
Direct Electron Detector (DED) (e.g., Gatan K3, Falcon 4) Camera that counts individual electrons with high quantum efficiency and fast readout. Essential for high-resolution SPA and low-dose, high-quality tilt-series collection.
Platinum Organometallic Precursor Used in the cryo-FIB/SEM to deposit a conductive, protective layer on the sample surface prior to milling, preventing charging and curtaining artifacts.

Application Notes

Cryo-Electron Tomography (Cryo-ET) occupies a distinct niche in structural biology, defined by the trade-off between biological context and resolution. This is central to research on host-virus interactions, where understanding the spatial and temporal context of infection is often as critical as obtaining atomic models of individual components. The following notes delineate this niche, with data derived from recent literature (2022-2024).

Table 1: Comparison of Cryo-ET Contextual Imaging vs. Single-Particle Analysis (SPA) for Purified Targets

Aspect Cryo-ET for Contextual Imaging (in situ) Cryo-ET/SPA for Purified Targets
Primary Objective Visualize macromolecular architecture in native cellular context. Determine high-resolution structure of isolated, homogeneous complexes.
Typical Resolution 2-4 nm (20-40 Å); locally up to ~1 nm (10 Å) with sub-tomogram averaging. 0.3-0.6 nm (3-6 Å) for SPA; 0.8-1.5 nm (8-15 Å) for tomograms of purified targets.
Sample Preparation Cryo-focused ion beam (cryo-FIB) milling of vitrified cells or organelles. Purification of target complex, followed by standard vitrification on EM grids.
Key Strength Reveals unbiased spatial relationships, stoichiometry, and conformational states of multiple complexes in a functional environment (e.g., virus budding from membrane). Achieves atomic or near-atomic detail for mechanistic understanding, drug docking, and precise mutagenesis studies.
Limitation Resolution is limited by sample thickness, molecular crowding, and radiation damage. Target density may be low. Biological context is lost. Functional states may be altered or missed during purification.
Key Output for Host-Virus Research Maps of viral entry, assembly, and egress sites; host organelle remodeling; immune synapse architecture. Atomic models of viral spike proteins, polymerase complexes, or host receptor-virus ligand complexes.

Table 2: Recent (2022-2024) Representative Studies Illustrating the Niche

Study Focus Technique Used Resolution Achieved Key Contextual Insight vs. Atomic Detail
HIV-1 assembly in CD4+ T cells in situ Cryo-ET ~18 Å (sub-tomogram average) Revealed Gag lattice organization at the plasma membrane and proximity to host ESCRT machinery.
SARS-CoV-2 spike dynamics SPA Cryo-EM 2.8-3.5 Å Defined atomic details of receptor-binding domain (RBD) "up" and "down" conformations in isolated protein.
Herpesvirus nuclear egress in situ Cryo-ET (cryo-FIB) 2-3 nm (context), ~12 Å (average) Visualized the in nucleo formation of nascent capsids and their docking at nuclear pore complexes.
Bacterial phage infection in situ Cryo-ET 3-4 nm Captured the moment of phage DNA injection into the host cytoplasm, showing host membrane deformation.

Protocols

Protocol 1: In Situ Cryo-ET Workflow for Imaging Virus-Infected Cells

Objective: To capture the structural context of viral infection within host cells.

  • Cell Culture and Infection: Grow relevant host cells (e.g., HAP1, HEK293) on glow-discharged gold EM grids. Infect with virus at a high MOI. Synchronize infection if needed.
  • Vitrification: At the desired time post-infection, blot and plunge-freeze the grid in liquid ethane using a vitrification device (e.g., Vitrobot). Maintain >95% humidity.
  • Cryo-Focused Ion Beam (Cryo-FIB) Milling:
    • Transfer grid to a cryo-FIB/SEM microscope under constant LN2 cooling.
    • Sputter-coat the sample with organometallic platinum to enhance conductivity.
    • Use a gallium ion beam to mill away excess cellular material, creating a ~100-200 nm thin lamella of the infected cell.
  • Cryo-Electron Tomography:
    • Insert the lamella into a 300 keV cryo-TEM.
    • Acquire a tilt series from -60° to +60° with a 2-3° increment, using dose-symmetric scheme. Total dose kept below ~100 e⁻/Ų.
    • Use a phase plate or Volta phase plate if available to enhance contrast.
  • Tomogram Reconstruction & Analysis: Align tilt series using patch-tracking algorithms (e.g., in IMOD). Reconstruct tomogram via weighted back-projection or SIRT-like methods (e.g., in AreTomo, EMAN2). Segment and annotate structures (virions, membranes, filaments) manually or using machine learning (EMAN2, Dynamo).

Protocol 2: Cryo-ET of Purified Viral Complexes for Intermediate-Resolution Analysis

Objective: To determine the architecture of a heterogeneous or fragile viral complex that is not amenable to high-resolution SPA.

  • Complex Purification: Express and purify the target complex (e.g., viral envelope glycoprotein array, nucleocapsid). Use gentle detergents and/or nanodiscs to maintain integrity. Confirm homogeneity by SEC and negative stain EM.
  • Grid Preparation: Apply 3-4 µL of sample (≈0.5-1 mg/mL) to a freshly glow-discharged Quantifoil grid. Blot (3-4 seconds, blot force 0) and vitrify as in Protocol 1.
  • Screening and Data Collection:
    • Screen for suitable ice thickness and particle distribution at 200 keV.
    • Acquire tomographic tilt series as in Protocol 1, but with finer angular increments (1-2°) if targeting higher resolution. Use a smaller physical pixel size (e.g., 1-2 Å/pixel).
  • Sub-tomogram Averaging:
    • Pick particles from the tomograms (e.g., using Tomography in Scipion).
    • Align and average sub-volumes using iterative refinement in RELION or Dynamo.
    • Perform CTF refinement and dose-weighting to push resolution.

Visualizations

G Start Start: Research Question Decision Primary Need? Start->Decision A1 Contextual Understanding (e.g., infection site, assembly pathway) Decision->A1 Yes A2 Atomic Mechanism (e.g., drug binding, conformational change) Decision->A2 No P1 Cryo-ET In Situ Workflow A1->P1 P2 SPA / Cryo-ET Purified Workflow A2->P2 O1 Output: 3D Tomogram (2-4 nm resolution) Spatial Maps, Architecture P1->O1 O2 Output: Atomic Model (0.3-0.6 nm resolution) Precise Coordinates, Drug Docking P2->O2 Synth Synthesis for Holistic Model O1->Synth O2->Synth

Title: Decision Tree for Cryo-ET vs. SPA in Host-Virus Research

G cluster_in_situ In Situ Cryo-ET Path cluster_purified Purified Target Path InfCell Virus-Infected Cell on EM Grid Vit Plunge Freezing (Vitrification) InfCell->Vit Mill Cryo-FIB Milling (Produce ~150nm Lamella) Vit->Mill TS Tilt-Series Acquisition in Cryo-TEM Mill->TS Recon Tomogram Reconstruction TS->Recon Out1 Contextual 3D Map Virus in Native Cell Recon->Out1 Purif Purify Viral Complex (e.g., Spike Protein) Vit2 Plunge Freezing (Vitrification) Purif->Vit2 TS2 Tilt-Series Acquisition or Single-Particle Movies Vit2->TS2 STA Sub-tomogram or Particle Alignment/Averaging TS2->STA Out2 High-Resolution Structure STA->Out2

Title: Parallel Workflows for Contextual and Atomic-Resolution Imaging

The Scientist's Toolkit

Table 3: Key Research Reagent Solutions for Host-Virus Cryo-ET

Item Function in Contextual (in situ) Studies Function in Purified Target Studies
Gold R2/2 or R2/1 Quantifoil Grids Provide conductive, flat support for cell growth and subsequent cryo-FIB milling. Standard support film for applying purified protein solutions.
Cryo-FIB Lift-Out Tools (e.g., AutoGrids) Enable precise transfer of a milled lamella to a TEM holder for tomography. Not typically used.
Liquid Ethane Propane Mix Cryogen for achieving ultra-fast vitrification, preserving native cellular state. Identical function: vitrifies purified samples in amorphous ice.
Fiducial Gold Beads (e.g., 10nm Protein A-Gold) Added to sample prior to tilt-series acquisition to aid in image alignment. Used in both workflows for tomographic alignment.
GraFix (Gradient Fixation) Reagents Less common for in situ, but can be used to stabilize large cellular complexes. Critical: Stabilizes large, fragile complexes (e.g., ribosomes, polymerases) prior to purification.
Nanodiscs (MSP, Saposin) Can be used to present host membrane receptors in a native-like lipid environment. Used to reconstitute membrane proteins (viral envelopes, ion channels) for structural studies.
Cryo-ET Software Suite (IMOD, Dynamo, RELION) For tomogram reconstruction, segmentation, and sub-tomogram averaging of cellular data. For sub-tomogram averaging and classification of purified, but heterogeneous, complexes.

Within the thesis on Cryo-Electron Tomography (Cryo-ET) for imaging host-virus interactions, Correlative Light and Electron Microscopy (CLEM) is an indispensable strategy. It bridges the functional, dynamic information from light microscopy with the high-resolution structural context of Cryo-ET. This integration enables targeted imaging of rare or transient cellular events, such as viral entry, replication organelle formation, or immune synapse evasion, within the native, hydrated cellular environment.

Key Application Areas:

  • Targeting Rare Events: Fluorescent tagging of viral components (e.g., capsid proteins, genomes) or host factors (e.g., receptors, innate immune sensors) allows for the identification and relocation of specific infection stages within a vitrified sample for subsequent Cryo-ET.
  • Contextualizing Molecular Dynamics: Live-cell imaging of fluorescent reporters (e.g., calcium flux, membrane fusion reporters) can be correlated with ultrastructural snapshots from Cryo-ET, linking dynamics to morphology.
  • Validating Labels in Situ: Fluorescent signals from antibody tags or expressed fluorescent proteins guide the precise milling and tomography of the labeled structure, confirming identity at the nanoscale.

Experimental Protocols

Protocol A: Sample Preparation for Cryo-CLEM of Virus-Infected Cells

This protocol details preparing a grid for targeted Cryo-ET of a fluorescently labeled viral infection event.

Materials: See "The Scientist's Toolkit" below. Procedure:

  • Cell Seeding & Infection: Seed mammalian cells (e.g., Hela or Vero E6) expressing a fluorescent marker (e.g., GFP-tagged viral protein, or a stain for organelles like ER-Tracker) onto gold EM grids (200-300 mesh, R2/2 Quantifoil) placed in a culture dish. Allow cells to adhere (4-6 hrs). Infect cells with virus at a low MOI (e.g., 0.1-1) to achieve sparse infection for easy targeting.
  • Fluorescent Imaging: At the desired post-infection time, transfer the grid to a cryo-light microscope stage. Using a 63x air or long-working-distance objective, acquire wide-field fluorescence and differential interference contrast (DIC) maps of the entire grid. Record stage coordinates for cells of interest (e.g., showing strong fluorescent puncta).
  • Vitrification: Immediately after imaging, blot the grid from the back side for 3-5 seconds and plunge-freeze it in liquid ethane using a vitrification device (e.g., Leica EM GP, Thermo Fisher Vitrobot). Maintain high humidity (>90%) to prevent evaporation.
  • Grid Transfer & Storage: Clip the grid under liquid nitrogen and store it in a cryo-storage box immersed in liquid nitrogen.

Protocol B: Correlation and Targeted Cryo-FIB Milling

This protocol covers the relocation of targets and preparation of lamellae for Cryo-ET.

Procedure:

  • Cryo-Fluorescence Relocation: Load the vitrified grid into a cryo-fluorescence microscope (e.g., Linkam CMS196, CryoSOLUTIONS cryo-CLEM stage). Cool the stage to -180°C. Use the recorded stage coordinates to relocate the cells of interest. Acquire high-resolution, high-sensitivity fluorescence images (using cameras like sCMOS or EMCCD) of the target areas.
  • Coordinate Transfer: Export the fluorescence overlay images and the precise stage/loader coordinates of the targets.
  • Loading into Cryo-FIB/SEM: Transfer the grid to a Cryo-FIB/SEM (e.g., Thermo Scientific Aquilos 2, Zeiss Crossbeam) using a cryo-transfer shuttle, maintaining cryogenic conditions.
  • Rough Correlation: In the FIB/SEM, use the grid geometry and low-mag SEM overview to perform a rough correlation with the cryo-fluorescence map.
  • Precise Lamella Targeting: Apply a conductive metal (platinum or gold) coating via GIS to protect the surface. Use the fluorescent signal map to navigate the SEM stage. Deposit a protective organometallic platinum layer (e.g., via gas injection system) directly above the fluorescent region of interest. Mill lamellae (150-200 nm thick) using the gallium ion beam, targeting the area directly beneath the deposited platinum stripe.
  • Lamella Quality Check: Image the lamella at various tilts (e.g., ±10°) in the SEM to assess thickness and integrity.

Protocol C: Cryo-Electron Tomography Data Acquisition

Procedure:

  • Transfer to Cryo-TEM: Transfer the lamella to a Cryo-TEM (e.g., Thermo Scientific Krios, Glacios) using a cryo-holder.
  • Low-Mag Relocation: At low magnification (e.g., 500x), use the lamella shape and fiducial markers (gold beads, if added during sample prep) to relocate the target area.
  • Tilt Series Acquisition: Using tomography software (e.g., SerialEM, Tomo), acquire a tilt series typically from -60° to +60° with a 2-3° increment, at a defocus of -6 to -10 µm, and a pixel size of 3-5 Å. Use a dose-symmetric scheme. Total dose should be kept below 80-100 e⁻/Ų.
  • Reconstruction & Analysis: Reconstruct the tomogram using weighted back-projection or SIRT-like algorithms (e.g., in IMOD, AreTomo). Segment and analyze structures of interest (e.g., viral particles, cytoskeletal elements, membranes) using software like EMAN2, Dynamo, or Amira.

Data Presentation

Table 1: Comparison of Cryo-CLEM Modalities for Host-Virus Studies

Modality Spatial Resolution Temporal Resolution Key Application in Host-Virus Research Primary Limitation
Live-cell CLEM -> Cryo-ET LM: ~250 nm; ET: ~2-4 nm Minutes to hours (pre-vitrification) Tracking viral particle motion leading to a static ultrastructural snapshot. Phototoxicity, fiducial drift during transfer.
Cryo-Fluorescence -> Cryo-ET LM: ~300-400 nm; ET: ~2-4 nm Fixed time point (post-vitrification) Precise targeting of fluorescently tagged viral proteins for tomography. Lower fluorescence signal at cryogenic temperatures.
Cryo-FLM -> Cryo-FIB -> Cryo-ET LM: ~300-400 nm; FIB: ~5 nm; ET: ~2-4 nm Fixed time point Targeting specific fluorescent cells for in-situ lamella preparation (most common workflow). Complex correlation workflow; risk of milling away the target.

Table 2: Typical Tomography Acquisition Parameters for Viral Structures

Parameter Value/Range Rationale
Accelerating Voltage 200-300 kV Optimal for penetration and contrast in thick lamellae.
Pixel Size 3.5 - 5.0 Å Balances field of view with resolution to visualize viral spikes/capsids.
Tilt Range ±60° Maximizes information while minimizing missing wedge effects.
Tilt Increment 2° or 3° Provides sufficient angular sampling for reconstruction.
Total Dose 80 - 100 e⁻/Ų Limits radiation damage to sensitive biological structures.
Defocus -6 to -10 µm Provides phase contrast for cellular and viral membranes.

Mandatory Visualization

G cluster_live Live-Cell Imaging (at 37°C) cluster_cryo Cryo-Preparation & Targeting cluster_lamella Lamella Preparation cluster_et High-Resolution Structure LM Wide-field/Confocal Fluorescence Imaging Target Identify & Map Fluorescent Target (e.g., viral entry) LM->Target Vit Rapid Vitrification (Plunge-freezing) Target->Vit Immediate Transfer cFLM Cryo-Fluorescence Microscopy (cFLM) Vit->cFLM CorrMap Generate Correlation Map cFLM->CorrMap FIB Cryo-Focused Ion Beam (Cryo-FIB) Milling CorrMap->FIB Coordinate Transfer Lamella Thin Lamella (∼200 nm) at Target FIB->Lamella CryoET Cryo-Electron Tomography Lamella->CryoET Cryo-Transfer Recon 3D Reconstruction & Analysis CryoET->Recon

CLEM to Cryo-ET Workflow for Viral Imaging

G Virus Viral Particle (Fluorescently Labeled) HostReceptor Host Cell Receptor (e.g., ACE2, CD4) Virus->HostReceptor 1. Attachment & Entry Cytoskeleton Cytoskeletal Remodeling Virus->Cytoskeleton 4. Cytoplasmic Transport Endosome Early/Late Endosome HostReceptor->Endosome 2. Endocytosis & Trafficking Endosome->Virus 3. Membrane Fusion & Uncoating VRC Viral Replication Complex (VRC) Cytoskeleton->VRC 5. VRC Formation & Genome Replication ER ER & Golgi Membranes VRC->ER 6. Assembly & Maturation ER->Virus 7. Egress

Viral Lifecycle Stages for CLEM Targeting

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Cryo-CLEM of Host-Virus Interactions

Item Function & Rationale Example Product/Type
Quantifoil Gold Grids (R2/2, 200 mesh) EM support grid. The gold is non-reactive, and the R2/2 hole pattern provides optimal ice thickness for tomography. Quantifoil Au 200 mesh R2/2
Fluorescent Viral Construct Enables live-cell tracking and cryo-targeting of specific viral components (capsid, envelope, polymerase). GFP-tagged HIV Gag, mNeonGreen-SARS-CoV-2 N protein.
Organelle-Specific Live-Cell Dyes Highlights host structures (ER, mitochondria, endosomes) for contextual targeting. ER-Tracker Red, MitoTracker Deep Red, LysoTracker.
Cryo-Plunger with Humidity Control For reproducible vitrification without sample evaporation, preserving native state. Thermo Fisher Vitrobot Mark IV, Leica EM GP2.
Cryo-Fluorescence Microscope Stage Enables high-resolution fluorescence imaging of samples at cryogenic temperatures. Linkam CMS196, CryoSOLUTIONS CLEM stage.
Gas Injection System (GIS) Precursors Deposits conductive metal (Pt) and protective organometallic layers for FIB milling. Platinum GIS (e.g., Trimethyl(methylcyclopentadienyl)platinum(IV)), E-beam Platinum.
Fiducial Markers for Correlation High-contrast markers for aligning light and electron microscopy images. TetraSpeck microspheres (for pre-vitrification), 10 nm colloidal gold beads.
Cryo-TEM Direct Electron Detector High-sensitivity camera for recording tomographic tilt series with low electron dose. Gatan K3, Falcon 4.

Within the thesis on Cryo-Electron Tomography (cryo-ET) for imaging host-virus interactions, structural models derived from tomograms represent hypotheses. These hypotheses—such as the molecular architecture of a viral fusion pore or the configuration of a host restriction factor bound to a viral capsid—require rigorous validation. Cross-checking with independent biochemical and mutational data is paramount to transform tomographic reconstructions into biologically reliable mechanistic insights, a critical step for downstream drug development targeting these interfaces.

Application Notes: Integrating Data Streams

The integration of cryo-ET with orthogonal methods creates a powerful validation cycle. The following table summarizes key correlative data types and their validation outputs.

Table 1: Cross-Validation Data Types and Interpretations

Validation Method Data Type How it Validates Cryo-ET Structure Expected Outcome for a Valid Model
Mutational Analysis (Alanine Scanning) Quantitative (Binding affinity, Infectivity IC₅₀) Disrupting an interface residue observed in cryo-ET should impair function. Mutations at predicted contact residues cause significant functional loss (>10-fold change).
Crosslinking Mass Spectrometry (XL-MS) Distance restraints (Å) Crosslinks provide maximum distance constraints between residues. >90% of identified crosslinks are within the maximum allowable distance in the cryo-ET model.
Surface Plasmon Resonance (SPR) Kinetic constants (kₒₙ, kₒff, K_D) Measures binding affinity of complexes visualized in situ. Measured K_D aligns with predicted stability from buried surface area in the structure.
Fluorescence Resonance Energy Transfer (FRET) Distance & proximity (10-100 Å) Provides in-solution distance ranges between labeled sites. FRET efficiency-derived distances are consistent with inter-label distances in the flexible-fitted model.
Cryogenic Fluorescence Microscopy (Cryo-FM) Co-localization data Targets and locates fluorescently labeled components within the tomogram. Fluorescence signal co-localizes precisely with the density attributed to the labeled component.

Detailed Experimental Protocols

Protocol 3.1: Coupling In-Situ Mutagenesis with Cryo-ET Workflow

Objective: To validate a putative protein-protein interface in a host-virus complex observed by sub-tomogram averaging.

  • Design: Based on the cryo-ET model, select 3-5 candidate interface residues on the host protein for alanine substitution.
  • Mutagenesis & Expression: Generate mutants using site-directed mutagenesis kits (e.g., Q5). Express and purify wild-type (WT) and mutant proteins.
  • Biochemical Assay: Perform a pull-down or SPR assay with the viral target protein. Quantify binding affinity relative to WT.
  • Functional Assay: For a relevant virus, perform a single-cycle infectivity assay in cells expressing the mutant host protein. Calculate IC₅₀.
  • Cryo-ET Sample Prep: Reconstitute the complex using the mutant protein under identical conditions to the WT. Prepare cryo-ET grids (e.g., Quantifoil R2/2).
  • Imaging & Analysis: Acquire tomograms and perform sub-tomogram averaging. Compare the mutant structure (local resolution map) to the WT.
  • Validation Criteria: A true interface mutant will show (a) >70% reduction in biochemical binding, (b) significant loss of viral infectivity, and (c) either a complete absence of the complex in tomograms or a distorted/destabilized density at the interface.

Protocol 3.2: Integrative Modeling with XL-MS Distance Restraints

Objective: To use XL-MS data to validate and refine a flexible-fitted atomic model into a cryo-ET envelope.

  • Sample Preparation: Form the native complex. Apply a membrane-permeable, MS-cleavable crosslinker (e.g., DSSO) at physiological pH.
  • XL-MS Analysis: Digest the complex, perform LC-MS/MS, and identify crosslinked peptides using software (e.g., XlinkX, pLink). Export a list of residue pairs and associated crosslinker lengths.
  • Distance Restraint File Generation: Convert each crosslink into an upper-bound distance restraint (typically Cα-Cα + 20 Å). Format for integrative modeling software (e.g., HADDOCK, IMP).
  • Integrative Modeling: Docked the atomic structures of components into the cryo-ET density using flexible fitting (e.g., MDFF). Use the XL-MS distance restraints as constraints during molecular dynamics refinement.
  • Validation Metric: Calculate the satisfaction rate. A validated model will have >85% of XL-MS restraints satisfied (distance < restraint upper bound). Restraints violated by >10 Å indicate potential model error.

Visualizations

G A Initial Cryo-ET Hypothesis (Complex Density Map) C Data Integration & Model Refinement A->C Provides Spatial Constraints B Biochemical & Mutational Data B->C Provides Functional Constraints D Validated Atomic Model C->D Generates D->B Predicts New Mutants/Assays E Biological Insight & Drug Target ID D->E Informs

Title: Cryo-ET Validation & Refinement Cycle

workflow cluster_0 cluster_1 cluster_2 Start Identify Interface from Cryo-ET Map Design Design Point Mutations at Interface Start->Design Exp Express & Purify WT & Mutant Proteins Design->Exp Assay1 In Vitro Binding Assay (SPR/BLI) Exp->Assay1 Recon Reconstitute Complex for Cryo-ET Assay1->Recon FuncAssay Cell-Based Functional Assay (e.g., Infectivity) Assay1->FuncAssay Image Acquire Tomograms & Subtomogram Average Recon->Image Compare Compare 3D Density WT vs. Mutant Image->Compare Compare->FuncAssay Correlate Structure-Function Valid Validated Interaction Interface Compare->Valid FuncAssay->Recon

Title: Mutational Validation Workflow for Cryo-ET

The Scientist's Toolkit: Key Research Reagents & Solutions

Table 2: Essential Reagents for Cross-Validation Experiments

Reagent / Solution Function / Application Example Product / Note
Membrane-Permeable, MS-Cleavable Crosslinker (e.g., DSSO) Generates distance restraints for XL-MS within native cellular or complex environments. Thermo Fisher Scientific DSSO (Disuccinimidyl sulfoxide). Cleavability aids MS/MS identification.
Chromatography Columns for Complex Purification Isolate native host-virus complexes for biochemical assays and cryo-ET sample prep. Size-exclusion (Superose 6 Increase) and affinity (HisTrap, StrepTrap) columns.
SPR/BLI Sensor Chips Immobilize bait molecule for real-time, label-free kinetic analysis of complex formation. Cytiva Series S CM5 chips (SPR); Streptavidin (SA) tips for BLI (FortéBio).
Cryo-EM Grids (Holey Carbon) Support vitrified sample for cryo-ET imaging. Choice affects ice thickness and particle distribution. Quantifoil R2/2 (200 mesh, 2µm holes); UltraAufoil (gold).
Fluorescent Labels for Cryo-FM Enable correlation of light microscopy with cryo-ET. Must be bright and photostable at cryogenic temperatures. Janelia Fluor dyes (e.g., JF549); ATTO 488; Site-specific labeling via SNAP/CLIP-tags.
Software for Integrative Modeling Computationally combine cryo-ET density, atomic models, and restraint data. Scipion, EMAN2 (processing); ChimeraX, HADDOCK, IMP (fitting & modeling).

Application Notes

The integration of Cryo-Electron Tomography (Cryo-ET) with super-resolution microscopy and quantitative proteomics is revolutionizing the study of host-virus interactions. This multi-scale approach provides an unprecedented view, from the molecular architecture of viral invasion complexes within a native cellular context to the global proteomic changes they induce. For drug development, this synergy identifies not only high-resolution structural targets but also the broader cellular pathways for therapeutic intervention, enabling the design of both precision antivirals and host-directed therapies.

Key Application Insights:

  • Contextualizing High-Resolution Structures: Cryo-ET reveals the in-situ structures of viral fusion pores or capsid docking sites with host organelles. Super-resolution light microscopy (e.g., STED, PALM) maps the spatial distribution and dynamics of these events across entire cells, identifying hotspots for Cryo-ET lamella preparation.
  • From Structure to System: Proteomic profiling (e.g., LC-MS/MS) of the same cell population under infection quantifies changes in protein abundance, phosphorylation, and protein-protein interactions. This data informs Cryo-ET targets—e.g., identifying a key upregulated host factor prompts its localization and structural characterization within cryo-lamellae.
  • Validating and Bridging Findings: Correlative Light and Electron Microscopy (CLEM) is foundational. Fluorescently tagged viral components (imaged via super-resolution) guide precise milling and tomography of the same event, directly linking dynamic behavior to ultrastructure.
  • Drug Mechanism Elucidation: This pipeline can visualize how a drug candidate disrupts the formation of a specific viral assembly compartment (Cryo-ET) while quantifying its impact on viral protein synthesis and immune signaling pathways (proteomics).

Table 1: Comparative Analysis of Integrated Techniques in Host-Virus Research

Technique Resolution Throughput Key Output for Host-Virus Studies Primary Limitation
Cryo-ET (in-situ) ~3-5 nm (local); ~1-3 nm (subtomogram avg.) Low (10s-100s of cells/lamellae per session) 3D macromolecular complexes in native cellular environment (e.g., viral ribonucleoprotein complexes in cytoplasm). Limited field of view; requires thinning (FIB milling).
Super-Resolution Microscopy (e.g., STORM) ~20 nm (xy); ~50 nm (z) Medium (10s of cells per experiment) Nanoscale localization and co-distribution of viral and host proteins (e.g., viral glycoprotein clusters on membrane). Requires fluorescent labeling; not native-state.
Quantitative Proteomics (TMT-LC/MS/MS) N/A (Molecular) High (1000s of proteins from millions of cells) Global protein abundance changes, post-translational modifications, and interaction networks during infection. Population average; loses single-cell and spatial information.

Table 2: Example Multi-Omic Data from Integrated Study of SARS-CoV-2 Infected Cells

Data Type Experimental Condition Key Finding Quantitative Result Follow-up Cryo-ET Target
Proteomics (Phospho) Cells 24h post-infection vs. Mock Hyperphosphorylation of host proteins involved in vesicle trafficking (e.g., VPS35, RAB GTPases). >2-fold increase in phosphorylation at specific sites on VPS35. Structure of viral replication organelles and associated host vesicles.
Super-Res (STED) Labeled Spike protein & ER marker Enrichment of Spike in distinct ER-derived double-membrane vesicle (DMV) clusters. Cluster size: 200 ± 50 nm. Density increased 5x in infected cells. Lamella targeted to DMV clusters for subtomogram averaging of viral pores.
Cryo-ET FIB-milled lamella from above cell In-situ structure of DMV and interior viral dsRNA. Subtomogram average of DMV pore complex at ~12 Å resolution. N/A (Primary finding)

Experimental Protocols

Protocol 1: Correlative STED and Cryo-ET Workflow for Viral Entry Site Analysis

Objective: To locate and image the initial site of virus-host membrane fusion within a cell using fluorescence and subsequently resolve its ultrastructure by Cryo-ET.

Materials (Research Reagent Solutions):

  • Cell Line: Cultured mammalian cells (e.g., Vero E6 or Huh-7).
  • Virus: Virus of interest with envelope proteins labeled via CRISPR knock-in with a self-labeling tag (e.g., HaloTag) or using fluorescently labeled antibodies post-fixation.
  • Fluorescent Dyes/Probes: Janelia Fluor 646 HaloTag ligand for live-cell imaging; Cell Mask for plasma membrane staining.
  • Buffers: PBS, 4% PFA (Electron Microscopy grade), 0.1% Glutaraldehyde in 0.1M Phosphate Buffer.
  • Microscopy Substrate: Carbon-coated, glow-discharged gold EM grids (R2/2 Quantifoil).
  • Key Instruments: Confocal/STED microscope with environmental chamber; Cryo-plunger; Cryo-FIB/SEM with CLEM stage; 300 kV Cryo-TEM with tomographic holder.

Procedure:

  • Sample Preparation: Infect grid-grown cells at low MOI. At desired time point, incubate with HaloTag ligand (e.g., 100 nM, 15 min), then stain membrane (1:1000, 5 min).
  • Live-Cell STED Imaging: Image grid at 37°C, 5% CO₂. Acquire STED z-stacks to identify cells with distinct fluorescent viral puncta at the plasma membrane. Record precise stage coordinates.
  • Rapid Cryo-Fixation: Immediately vitrify the grid using a plunge freezer. Maintain cryogenic chain.
  • Cryo-CLEM Transfer: Transfer grid to cryo-FIB/SEM with CLEM capability. Relocate target cell using stored coordinates and low-dose SEM imaging.
  • Lamella Preparation: Apply a protective organometallic platinum layer. Mill a ~200 nm thick lamella encompassing the fluorescently identified viral entry site using the cryo-FIB.
  • Cryo-ET Data Acquisition: Acquire a tilt series (e.g., -60° to +60°, 2° increment) at 300 kV. Perform alignment and reconstruction using IMOD or similar software.
  • Analysis: Segment and model membranes and viral particles in the tomogram to characterize fusion intermediates (hemifusion, pore formation).

G A Seed cells on EM grid B Infect with tagged virus A->B C Live-cell STED imaging (Map viral entry sites) B->C D Rapid cryo-plunge freezing C->D E Cryo-FIB/SEM CLEM (Relocate cell, mill lamella) D->E F Cryo-ET data acquisition E->F G Tomogram reconstruction & 3D analysis of fusion site F->G

Diagram Title: Workflow for Correlative STED and Cryo-ET of Viral Entry

Protocol 2: Integrative Proteomics and Targeted Cryo-ET for Viral Assembly Sites

Objective: To use differential proteomics to identify a host protein complex recruited during viral assembly, then determine its structural role via targeted Cryo-ET.

Materials (Research Reagent Solutions):

  • Cell Culture & Infection: Appropriate host cells; synchronized high-titer viral infection.
  • Proteomics Reagents: Lysis buffer (8M Urea, 50mM TEAB, protease/phosphatase inhibitors); Trypsin/Lys-C mix; Tandem Mass Tag (TMT) 16-plex reagents.
  • Antibodies: Validated antibodies for immunolabeling (CLEM) or immuno-precipitation.
  • Cryo-ET Supplies: High-pressure freezer with 100 µm planchettes; Freeze-substitution medium (e.g., 0.1% UA in acetone); Cryo-ultramicrotome or FIB/SEM.
  • Mass Spectrometer: High-resolution LC-MS/MS system (e.g., Orbitrap Eclipse).

Procedure: Part A: Quantitative (Phospho)Proteomics:

  • Harvest infected and mock cells in triplicate at peak assembly (e.g., 18h p.i.). Lyse, reduce, alkylate, and digest proteins.
  • Label peptides with TMT reagents. Pool samples and fractionate by high-pH reverse-phase HPLC.
  • Analyze fractions by LC-MS/MS. Search data against host and viral databases.
  • Perform statistical analysis (e.g., limma) to identify significantly upregulated host proteins or phosphosites at assembly sites (e.g., cytoskeletal adaptors). Part B: Targeted Cryo-ET:
  • Based on proteomics, select a candidate host protein (e.g., an actin nucleator). Prepare cells for Cryo-ET as in Protocol 1, optionally with immunogold labeling for CLEM.
  • Target Cryo-FIB milling to regions of high virion budding density (visible in SEM).
  • Acquire tomograms of assembly sites (e.g., plasma membrane, viral factories).
  • Use template matching or subtomogram averaging to locate the candidate protein density within the viral assembly landscape relative to budding virions.

G P Quantitative Proteomics (Infected vs. Mock Cells) ID Candidate Host Protein Identified & Validated P->ID Data-Driven Target Selection T Targeted Sample Prep for Cryo-ET ID->T Hypothesized Role C Cryo-FIB & Cryo-ET of Assembly Sites T->C S Subtomogram Averaging & Model Integration C->S

Diagram Title: From Proteomic Target to Cryo-ET Structure

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagents for Integrated Multi-Scale Host-Virus Research

Item Category Function in Workflow Example Product/Type
Fluorescent Protein/Self-Labeling Tags Labeling Enables live-cell super-resolution tracking of viral or host proteins for CLEM targeting. HaloTag, SNAP-tag, mNeonGreen.
CLEM-Compatible Gold EM Grids Sample Support Provides substrate for cell growth, fiducials for correlation, and stability under beam. Quantifoil Au R2/2, 300 mesh.
Cryo-Plunge Freezer Sample Prep Vitrifies cellular samples to preserve native-state hydration and structure for Cryo-ET. Leica EM GP2, Gatan Cryo-Plunge 3.
Gas Injection System (GIS) for FIB Sample Prep Deposits a conductive, protective organometallic platinum layer on the sample prior to FIB milling. Pt-based precursor (e.g., C9H16Pt).
Tandem Mass Tag (TMT) Kits Proteomics Allows multiplexed, quantitative comparison of protein abundance across multiple conditions (e.g., time course). TMTpro 16-plex Reagent Set.
High-Resolution LC Column Proteomics Separates complex peptide mixtures prior to MS injection, increasing proteome depth. C18, 75µm x 25cm, 1.6µm beads.
Cryo-TEM Direct Electron Detector Cryo-ET Captures high-sensitivity, low-noise images during tilt series, crucial for high-resolution reconstruction. Gatan K3, Falcon 4.

This Application Note provides a framework for evaluating the infrastructure and computational resource requirements for a research program focused on applying Cryo-Electron Tomography (Cryo-ET) to host-virus interactions. The decision-making process must balance the high-end capabilities of the technique against accessibility and operational costs. The following tables summarize current quantitative data.

Table 1: Comparative Analysis of Cryo-ET Data Acquisition Pathways

Pathway Option Estimated Capital Cost (USD) Annual Operational Cost (USD) Data Output (TB/year) Throughput (Tomograms/week) Key Accessibility Factor
In-house 300kV FEG Cryo-TEM with Tomography $5M - $10M $500k - $1M+ 100-500 10-50 Lowest accessibility; requires dedicated facility & expert team.
Shared Institutional Facility $50k - $200k (Access Fees) $50k - $200k 50-200 5-25 Moderate; depends on local facility scheduling and expertise.
National/Remote User Facility $10k - $50k (Proposal & Fees) $10k - $50k 20-100 2-15 High for beamtime; competitive proposal process, remote operation possible.
Commercial Service Provider $5k - $30k per project Variable per project Project-dependent Project-dependent Highest for single projects; no capital investment, limited control over scheduling.

Table 2: Computational Resource Requirements for Cryo-ET Pipeline

Processing Stage Recommended Hardware Estimated Cloud Cost (USD/Tomogram)* Storage Needs (per Tomogram) Key Software (Open Source)
Pre-processing & Denoising High-CPU Instance (32+ cores) $2 - $5 50-100 GB IMOD, Warp, cryoCARE
Tomogram Reconstruction GPU Instance (1x High-end GPU) $3 - $8 10-20 GB IMOD, AreTomo, Protomo
Subtomogram Averaging (STA) Multi-GPU Cluster (4-8 GPUs) $20 - $100+ 200-500 GB RELION, M, emClarity
Analysis & Visualization Workstation with Mid-range GPU $1 - $3 5-10 GB ChimeraX, Dynamo, Matlab/Python

*Cloud costs are estimates based on AWS/GCP spot or reserved instance pricing for processing a single tilt series of ~100 images to a final STA map. Costs scale dramatically with iteration and dataset size.

Experimental Protocols

Protocol 2.1: Integrated Workflow for In-situ Host-Virus Imaging via Cryo-ET

Objective: To capture the structural details of viral entry or assembly within a host cell using Cryo-ET.

Materials:

  • Cultured host cells (e.g., mammalian cell line relevant to virus study).
  • Virus of interest (purified or cell culture supernatant).
  • Lacey carbon EM grids (Au or Rhodium, 200-300 mesh).
  • Vitrification device (e.g., Vitrobot Mark IV).
  • Liquid ethane/propane mixture.
  • Cryo-TEM with tomography holder (200-300 kV FEG).
  • Software: SerialEM, IMOD, Warp.

Methodology:

  • Grid Preparation: Plasma clean lacey carbon grids. Seed host cells directly onto grids and culture to appropriate confluence.
  • Infection: Infect cells on-grid with virus at a pre-optimized MOI. Incubate for the desired time window (e.g., for early entry or late assembly).
  • Vitrification: At the target time point, blot and plunge-freeze the grid into liquid ethane using the Vitrobot (100% humidity, blot force/time optimized for cell thickness).
  • Screening & Targeting: Load grid into Cryo-TEM. At low magnification, identify regions of interest (ROIs) with intact, well-spread cells suspected of infection.
  • Fiducial Application (Optional, for thicker cells): Apply 10nm colloidal gold fiducials to the grid post-vitrification, inside the cryo-holder.
  • Tilt-Series Acquisition: Using SerialEM, navigate to an ROI. Set a dose-symmetric tilt scheme (e.g., -60° to +60°, 2-3° increment). Use low-dose mode with a total cumulative dose of 80-150 e⁻/Ų. Autofocus and track at each tilt.
  • Data Transfer: Transfer the dose-fractionated tilt-series (.mrc or similar) to a high-performance computing storage system.

Protocol 2.2: High-Throughput Subtomogram Averaging Pipeline

Objective: To classify and average sub-volumes containing viral glycoproteins or host factors to achieve high-resolution structural insights.

Materials:

  • Reconstructed tomograms (from Protocol 2.1).
  • Computational Cluster with multiple high-end GPUs (e.g., NVIDIA A100).
  • Software: RELION (v4.0+), M, Dynamo, PyEM.

Methodology:

  • Preprocessing: In Warp, perform motion correction, CTF estimation, and tomogram reconstruction. Apply denoising (e.g., cryoCARE) to enhance SNR.
  • Template Creation: Generate an initial low-resolution template from a few manually picked particles or from a known homologous structure low-pass filtered to 40-60Å.
  • Initial Particle Picking: Use template matching in Dynamo or a blob picker in RELION to generate initial particle coordinates.
  • Extraction & Initial Alignment: Extract sub-volumes (~64-128 pixels box size). Perform an initial round of alignment and classification in RELION (3D auto-refine followed by 3D classification) to remove junk particles and align to a common reference.
  • Iterative Refinement: Run several cycles of high-resolution 3D auto-refinement with per-particle CTF refinement and Bayesian polishing. Apply a mask around the protein of interest.
  • Classification: Use focused 3D classification (with or without alignment) to isolate different conformational states of the glycoprotein or to separate bound/unbound host receptors.
  • Post-processing: Sharpen the final map using the RELION post-processor, calibrating the resolution via Fourier Shell Correlation (FSC=0.143).

Mandatory Visualizations

G Acquisition Acquisition Processing Processing Acquisition->Processing Raw Tilt-Series (1-2 TB) Analysis Analysis Processing->Analysis Refined Map (<1 GB) Cost Cost Cost->Acquisition Capital & Beamtime Cost->Processing Compute & Storage Cost->Analysis Personnel & Software

Cryo-ET Workflow & Cost Drivers

G Cell Cell Virus Virus Attachment Attachment Virus->Attachment Entry Entry Attachment->Entry CryoET CryoET Attachment->CryoET  Visualize Receptor Binding Replication Replication Entry->Replication Entry->CryoET  Membrane Fusion Pore Assembly Assembly Replication->Assembly Release Release Assembly->Release STA STA Assembly->STA  Glycoprotein Spike Structure Release->Virus

Host-Virus Cycle & Cryo-ET Insights

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Host-Virus Cryo-ET Studies

Item Function in Cryo-ET Workflow Example Product/Note
Lacey Carbon Grids (Au/Rh) Provide a support film with holes, allowing cells to span voids for optimal imaging without background noise. Quantifoil R2/2, Au 300 mesh. Rhodium grids reduce catalysis.
Cultured Cells (Low Background) Host cells must be thin (<500nm) and adherent for on-grid culture. EM-optimized cell lines (e.g., fibroblast-like).
Fiducial Gold Beads (10-15nm) Provide reference points for accurate alignment of tilt-series images during tomogram reconstruction. BSA-treated colloidal gold from Cytodiagnostics.
Vitrification System Rapidly plunges grids into cryogen to preserve cellular structures in a near-native, vitreous ice state. Thermo Fisher Vitrobot Mark IV or Leica GP2.
Cryo-TEM with Direct Detector High-voltage TEM equipped with a camera capable of counting individual electrons for low-dose imaging. 300kV Titan Krios with Gatan K3 or Falcon 4 detector.
Tomography Acquisition Software Automates the complex process of tilting, tracking, focusing, and image capture with minimal electron dose. SerialEM (most common), Tomography (Thermo Fisher).
Subtomogram Averaging Software Performs alignment, classification, and averaging of thousands of extracted sub-volumes to achieve high resolution. RELION (gold standard), M, emClarity.
High-Performance Computing (HPC) CPU/GPU clusters necessary for computationally intensive steps like tomogram reconstruction and STA. Local cluster or cloud-based (AWS ParallelCluster, Google Cloud).

Conclusion

Cryo-electron tomography has emerged as an indispensable, transformative tool for virology, providing an unprecedented, three-dimensional view of viruses interacting with their host cells in a near-native state. By mastering its foundational principles and complex methodology, researchers can move beyond static structures to dynamic processes, revealing the mechanistic details of viral entry, replication, assembly, and egress. While challenges in sample preparation, data acquisition, and processing persist, ongoing optimization and integration with complementary techniques continue to push the boundaries of what is possible. The future of cryo-ET lies in higher automation, more sophisticated AI-driven analysis, and tighter integration with functional assays, paving the way for a new era of rational, structure-guided antiviral drug and vaccine design that targets vulnerable stages of the viral life cycle within the cellular environment.