Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy
July 19th, 2021
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Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.
Overview
This article demonstrates the use of a sandwich freezing device (SFD) for rapid freezing of biological specimens, enabling superior preservation of cellular ultrastructure for electron microscopy. The protocol details specimen preparation, rapid freezing, freeze-substitution, resin embedding, ultrathin sectioning, staining, and imaging, applicable to bacteria, yeast, cultured cells, animal and human tissues, and viruses.
Key Study Components
Area of Science
- Cell biology
- Electron microscopy
- Sample preparation techniques
Background
- Chemical fixation often introduces artifacts and does not adequately preserve cellular ultrastructure.
- Rapid freezing, particularly sandwich freezing, prevents ice crystal formation and better maintains native cell structure.
- Handmade sandwich freezing devices limited broader adoption; a new commercially available device addresses this.
- Freeze-substitution and resin embedding are essential for preparing ultrathin sections for electron microscopy.
Purpose of Study
- To demonstrate the use of a commercial sandwich freezing device for rapid freezing of diverse biological specimens.
- To provide a detailed protocol for preparing specimens for electron microscopy after rapid freezing.
- To show the effectiveness of this method in preserving natural cellular morphology.
Methods Used
- Preparation of liquid propane and copper discs for freezing.
- Application of cell or tissue samples between copper discs and rapid freezing in solid propane using the SFD.
- Freeze-substitution in acetone with osmium tetroxide at -80°C, followed by gradual warming.
- Resin embedding, block trimming, ultrathin sectioning (50–70 nm), and mounting on copper grids.
- Staining with uranyl acetate and lead citrate, and imaging by electron microscopy.
Main Results
- Ultrathin sections of E. coli, S. cerevisiae, cultured cells, and human skin showed clear, natural morphology under electron microscopy.
- Hepatitis B virus core particles were successfully observed by cryo-electron microscopy after rapid freezing.
- Glutaraldehyde fixation prior to freezing enabled good preservation up to 200 µm depth, comparable to high-pressure freezing.
- Minimal ice crystal formation was achieved by applying very small sample volumes.
Conclusions
- The sandwich freezing device enables easy, low-cost, and effective preservation of cellular ultrastructure for electron microscopy.
- This method is broadly applicable to various biological specimens, including cells, tissues, and viruses.
- Proper sample handling and minimal volume application are critical for optimal results.
Frequently Asked Questions
What is the main advantage of sandwich freezing over chemical fixation?
Sandwich freezing prevents ice crystal formation and better preserves the natural ultrastructure of cells, avoiding artifacts and extraction of cell contents common with chemical fixation.
What types of specimens can be processed using the sandwich freezing device?
The device can be used for bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses.
How is freeze-substitution performed after rapid freezing?
Specimens are transferred to acetone containing osmium tetroxide at -80°C for 2–4 days, then gradually warmed to room temperature before resin embedding.
Why is it important to use a very small amount of sample on the copper discs?
Using a minimal sample volume prevents ice crystal formation during rapid freezing, ensuring optimal preservation of ultrastructure.
How does glutaraldehyde fixation improve tissue freezing?
Pre-fixing tissues with glutaraldehyde allows for good freezing and preservation of structure up to 200 micrometers deep, similar to high-pressure freezing methods.
What are the key steps after freeze-substitution for preparing samples for electron microscopy?
Key steps include resin embedding, block trimming, ultrathin sectioning, mounting on grids, staining with uranyl acetate and lead citrate, and imaging under an electron microscope.
What results can be expected using this protocol?
Researchers can expect clear, artifact-free images of cellular and tissue ultrastructure, with natural morphology preserved in a variety of biological specimens.
Executive Industry Relevance
Preserving native cellular ultrastructure is critical for mechanistic de-risking and target validation in early discovery and translational research. The sandwich freezing device enables rapid, artifact-minimized sample preparation for electron microscopy, supporting high-confidence morphological analysis across diverse biological systems. This capability enhances predictive confidence at key inflection points in the biopharma R&D pipeline.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables high-fidelity visualization of cellular and subcellular structures for mechanistic hypothesis testing.
- Reduces fixation artifacts, supporting functional target validation and biological de-risking.
- Facilitates comparative ultrastructural analysis across cell types and tissues for portfolio triage.
Screening & Assay Development
- Provides standardized, reproducible sample preparation for downstream EM-based assays.
- Supports quantitative morphological assessment for screening readiness and assay validation.
- Enables scalable preparation of diverse biological specimens, including bacteria, yeast, mammalian cells, and tissues.
Translational & Preclinical Research
- Aligns ultrastructural preservation with disease-relevant tissue analysis for translational continuity.
- Supports risk-adjusted advancement decisions by enabling robust morphological endpoints.
- Facilitates cross-species and cross-model system comparisons for preclinical validation.
Pipeline & Workflow Integration
The sandwich freezing device integrates into the discovery-to-preclinical continuum, enabling robust sample preparation for EM-based analysis from early discovery through translational research.
- Discovery Biology: Supports hypothesis-driven interrogation of cellular architecture and pathway localization.
- Screening: Delivers reproducible, quantitative ultrastructural readouts for assay development and compound evaluation.
- Analytics: Provides high-resolution morphological data for statistical comparison of experimental conditions.
- Translational Research: Maintains ultrastructural integrity in clinically relevant tissues, supporting biomarker alignment.
- Enterprise Reuse: Offers a standardized, scalable protocol adaptable across multiple biological models and research teams.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Standardizes sample preparation, improving reproducibility and scalability across projects.
- Strategic Value: Enables informed go/no-go decisions and reduces late-stage biological risk.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of discovery and translational programs.
Implementation Considerations
- Requires expertise in electron microscopy and sample handling for optimal results.
- Needs access to rapid freezing instrumentation, cryogens, and EM infrastructure.
- Demands cross-team standardization of sample preparation protocols for reproducibility.
- Adaptable to a wide range of biological specimens, including cells, tissues, and viruses.
- Sample volume and handling are critical to prevent ice crystal formation and preserve ultrastructure.
Biopharma FAQs
Why does null hypothesis testing matter for ultrastructural target validation?
Null hypothesis testing using ultrastructural data enables objective assessment of morphological differences between experimental and control groups, supporting robust target validation and reducing interpretive bias in early discovery.
How does independent variable isolation fit the sandwich freezing workflow?
Isolating variables such as fixation method or sample type within the sandwich freezing protocol allows teams to attribute observed ultrastructural changes directly to experimental interventions, strengthening mechanistic conclusions.
What do quantitative dependent variable measurements enable in EM analysis?
Quantitative measurements of ultrastructural features, such as organelle size or membrane integrity, enable statistical comparison across conditions and inform data-driven decisions in assay development and target validation.
Why are replication requirements critical for cross-functional EM studies?
Replication ensures that observed ultrastructural findings are reproducible and reliable, facilitating cross-functional collaboration and enabling consistent interpretation across discovery and translational teams.
What statistical analysis capabilities are needed before implementing EM-based workflows?
Teams must establish statistical frameworks for analyzing morphological data, including threshold setting and variance analysis, to ensure that EM-based findings support actionable R&D decisions and portfolio advancement.
