Overview
We provide sample preparation and imaging services for the study of biological ultrastructure. Cellular electron microscopy is widely used to visualize organelles within intact cells, bacterial cell walls, chloroplasts, membranes, tissues, and other cellular or subcellular structures.
The choice of preparation method is adapted to each sample type and scientific question in order to preserve the specimen as well as possible. In addition to classical morphological analysis, recent developments in cryo-FIB/SEM and cryo-electron tomography now make it possible to study cellular structures in a near-native state and to contribute to integrative structural biology.
The platform supports projects ranging from conventional room-temperature electron microscopy to cryogenic preparation, correlative microscopy, cryo-FIB/SEM lamella preparation, and cryo-electron tomography.
Main Applications
The platform offers expertise for the preparation and observation of biological samples, including:
- Ultrastructural analysis of cells, bacteria, tissues, organelles, membranes, and cell walls
- Conventional morphological analysis by transmission electron microscopy
- Surface imaging by scanning electron microscopy
- Immunolabelling to localize proteins or structures of interest
- Correlative light and electron microscopy
- Cryogenic preparation for improved structural preservation
- Cryo-FIB/SEM milling of thin lamellae from frozen samples
- Cryo-fluorescence imaging, cryo-TEM, and cryo-electron tomography after lamella preparation
For more info, see:
https://www.protocols.io/view/cryo-fixation-and-resin-embedding-of-biological-sa-bp2l62kndgqe/v1
Sample Preparation Approaches
Different preparation methods are available depending on the nature of the specimen, the level of preservation required, and the type of information sought.
Conventional Room-Temperature Electron Microscopy
Room-temperature preparation is suitable for many morphological studies on tissues, cell monolayers, bacteria, algae, and other biological samples.
This approach may include chemical fixation, resin embedding, ultrathin sectioning, staining, and observation by transmission electron microscopy. It provides access to the internal organization of cells and tissues, including organelles, membranes, bacterial envelopes, and other ultrastructural features.
Available embedding media include classical resins such as EPON and LR White.
Tokuyasu Cryo-Sectioning and Immunolabelling
The Tokuyasu technique is used when molecular localization is required. It allows immunolabelling on cryo-sections in order to detect proteins or structures of interest within cells, bacteria, algae, tissues, or other biological specimens.
This method can be combined with high-pressure freezing, freeze substitution, room-temperature sectioning, and manual or automated immunolabelling.
A successful experiment requires a clean sample and a suitable, validated antibody.
High-Pressure Freezing and Freeze Substitution
High-pressure freezing is used to improve the preservation of biological structures, especially for samples such as tissues, pellets, thicker cell samples, or concentrated specimens.
After freezing, samples can be processed by freeze substitution, embedded in resin, sectioned by ultramicrotomy, and imaged by transmission electron microscopy.
High-pressure freezing can also be followed by cryo-ultramicrotomy for CEMOVIS applications.
CEMOVIS
CEMOVIS, or cryo-electron microscopy of vitreous sections, allows the observation of vitrified biological material after cryo-sectioning. It is particularly demanding in terms of sample quality and requires pure, concentrated material.
Plunge Freezing for Cryo-Electron Microscopy
Plunge freezing, also known as cryo-plunge freezing or immersion freezing, is used for small samples such as cells or bacteria grown directly on electron microscopy grids.
This method preserves samples close to their native hydrated state and is especially suitable for cryo-electron microscopy. Plunge freezing can be performed using systems such as the Vitrobot or Leica EM-GP2.
Cryo-FIB/SEM
Cryo-FIB/SEM gives access to the internal organization of frozen, relatively thick biological samples while maintaining them close to their native state.
Cells are typically grown on gold grids and vitrified before milling. Fluorescence microscopy can be used before or after milling to locate a protein or region of interest. The cryo-FIB/SEM then produces a thin lamella that is transparent to electrons and suitable for further analysis by cryo-TEM or cryo-electron tomography.
This approach is particularly useful for in situ structural studies, where the objective is to observe macromolecular or cellular organization inside preserved cellular environments.
Imaging Techniques
The platform provides several imaging modalities for biological samples.
Transmission Electron Microscopy
Transmission Electron Microscopy is used to observe thin sections prepared by conventional ultramicrotomy, Tokuyasu cryo-sectioning, or cryogenic methods. It is the main imaging technique for the analysis of internal ultrastructure.
Scanning Electron Microscopy
Scanning Electron Microscopy is used to visualize the surface topology of biological samples. Observations can be performed at room temperature or under cryogenic conditions.
Cryo-Correlative Microscopy
Cryo-correlative microscopy allows regions or proteins of interest to be located in frozen samples, in collaboration with the photonic microscopy platform.
Imaging After Cryo-FIB/SEM Milling
After lamella preparation, several imaging options are available:
- Cryo-fluorescence imaging
- Conventional 2D cryo-TEM imaging
- Cryo-electron tomography
Choosing the Appropriate Preparation Method
The optimal preparation method depends on the sample type and the scientific objective.
| Sample or objective | Recommended preparation |
|---|---|
| Cell monolayers | Chemical fixation or plunge freezing, depending on the question |
| Bacteria grown on grids | Plunge freezing using Vitrobot or Leica EM-GP2 |
| Tissues | Chemical fixation or high-pressure freezing |
| Pellets | High-pressure freezing |
| Morphological analysis | Pure sample suitable for fixation, embedding, and sectioning |
| Immunolabelling | Pure sample and validated antibody |
| CEMOVIS | Pure and concentrated sample |
| Cryo-FIB/SEM | Cells grown on gold grids and vitrified before milling |
Sample Requirements and Handling
Biosafety
Samples must comply with the platform’s biosafety and biohazard regulations.
Accepted samples include:
- Biosafety level L1 samples
- Biosafety level L2 samples when chemically fixed
- BSL2 cells for cryo-FIB/SEM, when compatible with cryo-fixation procedures
GMO Compliance
Samples must comply with the relevant GMO regulations and institutional declarations.
Sample Return or Disposal
After the experiment, samples can be returned to the user upon request. Otherwise, they are stored for up to two weeks before being safely discarded.
Available Techniques
The platform combines a broad range of preparation and imaging methods for biological electron microscopy.
Fixation and Preservation
- Chemical fixation
- High-pressure freezing
- Plunge freezing
Embedding and Substitution
- Classical resin embedding, including EPON and LR White
- Freeze substitution
- Cryo-substitution and embedding
Sectioning
- Classical ultramicrotomy
- Tokuyasu cryo-sectioning
- Cryo-ultramicrotomy for CEMOVIS
Labelling and Staining
- Post-staining
- Manual immunolabelling
- Automated immunolabelling
Observation
- Transmission Electron Microscopy
- Scanning Electron Microscopy
- Cryo-electron microscopy
- Cryo-electron tomography
Instruments and Equipment
The platform is equipped with dedicated instruments for cellular electron microscopy sample preparation and imaging.
Cryogenic and Room-Temperature Sample Preparation
- Leica EM ICE high-pressure freezer
- Leica HPM100 high-pressure freezer
- Leica EM-GP2 plunge freezer
- Leica AFS2 and AFS1 freeze-substitution systems
- Leica AFS2 with FSP module
- Leica UC7 ultramicrotome
- Leica UC7/FC7 cryo-ultramicrotome
- Leica IGL immunolabelling system
Correlative microscopy developments are ongoing, in connection with complementary imaging facilities.
Dedicated Staff
The platform is supported by staff members with expertise in biological sample preparation, electron microscopy, cryogenic methods, and correlative approaches.
- Benoit Gallet
- Daphna Fenel
- Christine Moriscot
- Guy Schoehn
Location
The platform is located at IBS, on the ground and first floors, within the Methods and Electron Microscopy Group.
Quality Certification
The platform is certified according to ISO 9001 and NF X 50-900 quality standards.