Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
November 20th, 2021
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We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the Saccharomyces cerevisiae.
Overview
This article presents a detailed protocol for preparing Saccharomyces cerevisiae (yeast) samples for cryo-electron tomography (cryo-ET) using cryo-focused ion beam milling (cryo-FIBM). The workflow covers all steps from yeast cultivation and vitrification to lamella preparation and data acquisition, enabling high-resolution structural studies of cellular interiors.
Key Study Components
Area of Science
- Structural biology
- Cellular and molecular biology
- Cryo-electron microscopy
Background
- Cryo-ET provides near-atomic resolution structural data of macromolecular complexes in situ.
- High-resolution cryo-ET is limited to thin specimens (<200 nm), restricting studies to cell peripheries.
- Cryo-FIBM enables preparation of thin cellular lamellae from thicker samples, allowing access to cell interiors.
- Saccharomyces cerevisiae is a widely used eukaryotic model organism in biological research.
Purpose of Study
- To provide a step-by-step protocol for preparing yeast lamellae suitable for cryo-ET.
- To demonstrate vitrification and lamella preparation for both isolated cell patches and monolayers on TEM grids.
- To facilitate high-resolution structural studies of eukaryotic cells beyond peripheral regions.
Methods Used
- Yeast cultivation in sterile conditions to exponential phase.
- Preparation and plasma cleaning of EM grids.
- Vitrification of yeast suspensions on grids by plunge freezing in liquid ethane using a Vitrobot.
- Mounting vitrified grids into cartridges and transferring to a dual-beam FIB/SEM microscope with cryo-stage.
- Protective coating of samples with organic and inorganic layers.
- Focused ion beam milling to produce thin lamellae (<250 nm) from cell clusters or monolayers.
- Transfer of milled lamellae to a cryo-TEM for tomography data acquisition.
- Data processing using Etomo and Amira software.
Main Results
- Efficient preparation of yeast lamellae suitable for cryo-ET analysis.
- Protocols for both cell clusters and monolayer samples, with discussion of their respective advantages and disadvantages.
- Production of lamellae thin enough for electron beam transmission and high-resolution imaging.
- Successful acquisition and processing of cryo-ET data from prepared lamellae.
Conclusions
- The described protocol enables reliable preparation of yeast samples for in situ structural studies using cryo-ET.
- Cryo-FIBM extends the applicability of cryo-ET to the interior of eukaryotic cells.
- This workflow supports advanced cellular and molecular investigations in structural biology.
Frequently Asked Questions
What is the main advantage of using cryo-focused ion beam milling (cryo-FIBM) in cryo-ET sample preparation?
Cryo-FIBM allows the preparation of thin lamellae from thick cellular samples, enabling high-resolution cryo-ET imaging of cell interiors that are otherwise inaccessible due to sample thickness limitations.
Why is Saccharomyces cerevisiae used as a model organism in this protocol?
Saccharomyces cerevisiae is a well-studied eukaryotic model organism with broad applications in cellular and molecular biology, making it ideal for demonstrating protocols applicable to other eukaryotic cells.
How are yeast cells vitrified for cryo-ET analysis?
Yeast cell suspensions are applied to plasma-cleaned EM grids and rapidly plunge-frozen into liquid ethane using a Vitrobot, preserving cellular structures in a near-native state.
What are the key steps in preparing lamellae for cryo-ET?
Key steps include cultivating yeast, vitrifying cells on EM grids, mounting grids in cartridges, coating with protective layers, milling thin lamellae using a focused ion beam, and transferring lamellae to a cryo-TEM for imaging.
How is sample contamination prevented during the workflow?
Sterile techniques are used during cultivation, and all transfers and manipulations are performed under cryogenic conditions with liquid nitrogen to prevent ice contamination and preserve sample integrity.
What software is used for processing cryo-ET data in this protocol?
Etomo is used for tomogram reconstruction, and Amira is used for segmentation and further analysis of the acquired data.
What are the differences between preparing lamellae from cell clusters versus monolayers?
Both approaches are described; monolayers may offer more uniform lamellae, while clusters can provide access to different cellular regions. The protocol discusses the pros and cons of each sample type.
Executive Industry Relevance
Cryo-focused ion beam micromachining (cryo-FIB) enables high-resolution cryo-electron tomography (cryo-ET) of eukaryotic cell interiors, overcoming traditional thickness limitations. This workflow supports structural biology teams seeking predictive confidence in macromolecular complex localization and organization within intact cellular environments. The protocol's reproducibility and scalability position it as a foundational capability for early discovery and translational research pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct visualization of macromolecular complexes in native cellular context for mechanistic de-risking.
- Supports functional target validation by revealing spatial relationships and subcellular localization.
- Facilitates hypothesis-driven interrogation of eukaryotic cell architecture relevant to disease models.
Screening & Assay Development
- Provides standardized preparation of vitrified cellular lamellae for downstream imaging workflows.
- Ensures reproducible sample thickness and quality, critical for quantitative structural assays.
- Enables reliable comparison of cellular states or perturbations in screening campaigns.
Translational & Preclinical Research
- Aligns structural insights with disease-relevant cellular models such as Saccharomyces cerevisiae.
- Supports continuity from discovery through preclinical validation by enabling in situ analysis of cellular machinery.
- Reduces translational risk by providing high-fidelity structural data for biomarker or target assessment.
Pipeline & Workflow Integration
This protocol integrates from early discovery through lead identification and preclinical research, providing a bridge between molecular hypothesis and cellular validation.
- Discovery Biology: Advances hypothesis testing by enabling direct structural interrogation of cellular interiors.
- Screening: Delivers reproducible lamellae suitable for high-throughput or comparative imaging studies.
- Analytics: Generates quantitative tomographic data for robust statistical analysis and condition comparison.
- Translational Research: Maintains continuity by supporting disease-relevant model systems and biomarker alignment.
- Enterprise Reuse: Establishes a reusable workflow adaptable to diverse eukaryotic cell types and research questions.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Standardizes sample preparation and imaging, enhancing reproducibility and scalability.
- Strategic Value: Improves go/no-go decision-making and capital efficiency by providing high-content structural data early in the pipeline.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of structurally validated targets.
Implementation Considerations
- Requires expertise in cryo-EM, FIB/SEM operation, and sample vitrification.
- Demands access to specialized instrumentation and cryogenic infrastructure.
- Necessitates rigorous cross-team standardization for reproducible lamella preparation.
- Adaptable to various eukaryotic models but may require protocol optimization for different cell types.
- Sample thickness and contamination control are critical for successful downstream imaging.
Biopharma FAQs
Why does null hypothesis testing matter for cryo-ET target validation?
Null hypothesis testing in cryo-ET enables objective assessment of whether observed macromolecular structures differ significantly from controls, supporting robust target validation and reducing false positives in early discovery.
How does independent variable isolation fit the cryo-FIB workflow?
Isolating variables such as sample thickness and vitrification conditions ensures that structural differences observed in cryo-ET are attributable to biological factors, not technical artifacts, strengthening discovery-stage conclusions.
What do quantitative lamella thickness measurements enable in this protocol?
Quantitative measurement of lamella thickness ensures samples are within the optimal range for electron transmission, enabling high-resolution imaging and reliable comparison across experimental conditions.
Why are replication requirements critical for cross-functional collaboration?
Replication of lamella preparation and imaging protocols ensures that structural findings are reproducible across teams, facilitating data integration and collaborative decision-making in multi-disciplinary R&D environments.
What statistical analysis capabilities are required before cryo-ET implementation?
Robust statistical analysis of tomographic data, including thickness distribution and structural feature quantification, is essential for validating findings and informing go/no-go decisions in the biopharma pipeline.
