- Negative staining
- Cellular Electron Microscopy
- Morphological analysis
- Cryo-FIB/SEM
- Single Particle cryo-Electron Microscopy (SPA cryo-EM)
- Cryo-Electron Tomography (cryo-ET)
- CryoFIB/SEM milling
- Microcrystal Electron Diffraction (MicroED)
Sample Requirement and Handling
Presentation
The Electron Microscopy (EM) Platform provides high-quality morphological and structural information on biological samples, ranging from proteins and macromolecular complexes, to viruses and intact cells. Scientists from academia or industry are provided with access to state-of-the-art equipment for sample preparation and imaging by means of electron microscopy and can be introduced and supported in the use of such equipment.
The EM Platform offers traditional room temperature electron microscopy methods as well as cryogenic electron microscopy (cryo-EM), including negative staining, cellular EM, single particle analysis (SPA) and cryo-electron tomography (cryo-ET) experiments (Methodology). The facility houses 3 transmission electron microscopes (TEMs) and a dual beam Focused Ion Beam / Scanning Electron Microscope (FIB/SEM) (instruments). Its staff can provide service, technical support and expertise to ensure optimal and efficient utilization of the instruments and to assist researchers achieve their goals, as well as carrying out maintenance tasks and being involved in methodological developments in sample preparation, data acquisition or image analysis (Methodology).
Methodology
Depending on the nature of the sample and on the research objective we propose different experimental approaches (see the experiments section for details):
- Quality Control: Negative staining techniques for sample evaluation and quality assessment.
- Morphological Analysis: Negative staining and cellular electron microscopy to study the structural features of biological samples.
- Structural Analysis: Advanced techniques such as cryo-EM SPA, cryo-ET, micro-electron diffraction, and 3D cellular EM for detailed structural studies at medium to high-resolution.
In addition, the platform staff can support researchers that wish to access the facility in various ways. We can propose:
- Full Service: “A la carte” project support, from sample preparation to data analysis.
- Training: Hands-on training programs tailored to users’ needs, enabling proficiency in electron microscopy techniques.
- Instrument Access: Direct access to our state-of-the-art instruments for expert users.
This flexible approach ensures that all users, from beginners to experienced researchers, can effectively utilize our resources.
For each technique, details about sample requirement can be found in the “general guidelines of sample requirements” part
Negative staining (NS)
Quality control by negative staining
Negative staining techniques can provide valuable information about the quality of a biological sample. Typically, it is used to assess a sample’s homogeneity (purity, aggregation) and to determine its oligomerization state. The obtained images can also be used for single-particle analysis to 2D classification and low-resolution 3D reconstruction of a protein or complex. Quality control by NS is strongly recommended for samples intended to be used for structural studies and required prior to cryo-EM sample vitrification.
Morphological analysis by negative staining
Negative staining can also be used to perform a morphological analysis of the sample, such as size distribution of nano-particles, decoration of nano-particles or viruses etc. Basic image analysis can be performed.
The Tecnai T12 LaB6 120kV TEM equipped with an Orius 1000 camera (wide field) and a TVIPS F416 camera (4K x 4K) is primarily used for NS. Alternatively, the Tecnai F20 200 kV equipped with a CETA camera can also be used for NS. (Instruments)
Cellular Electron Microscopy (CEM)
For details see : https://www.protocols.io/view/cryo-fixation-and-resin-embedding-of-biological-sa-bp2l62kndgqe/v1
Cellular EM is commonly used for the study of ultrastructure: organelles within an intact cell, bacteria cell wall, chloroplast etc… A wide variety of different sample preparation techniques exists, each adapted to different types ensuring optimal preservation of the specimen.
In 2025, in addition to these morphological aspects, thanks to new instruments such as cryo-FIB/SEM coupled with cryo-electron tomography, cellular electron microscopy can also be used to carry out structural studies and is a key technique for integrative structural biology.
Morphological analysis
RT Sample prep techniques for CEM
Conventional chemical fixation for tissue samples and cell monolayers, plastic embedding, sectionning followed by staining. This enables access to the morphology of the sample (organite within the cell, cell wall etc..).
Tokuyasu technique : immunolabeling to locate structures of interest within a sample (bacteria, cell, algae etc..) after high pressure freezing, freeze substitution and room temperature sectioning.
Cryo Sample preparation techniques for CEM
Cryo methods will better preserve the different samples and allow structural analysis.
High pressure freezing, freeze substitution followed by ultramicrotomy or High pressure freezing followed by cryo-ultramicrotomy (CEMOVIS)
Plunge freezing (immersion freezing) for cryo-EM (“cryo plunge freezing”) for best possible preservation of small samples close to their native state. In this case, cells or bacteria are grown on grids:
Cryo-FIB/SEM
Get access to the native state of part of a “massive” sample. One can also locate the protein of interest using fluorescent microscopy before or after milling. The result is a thin lamella transparent to electrons which will be submitted to tomography.
The preparation is sample dependent:
- Cells or bacteria grown on grids: Vitrobot or Leica GP2 plunge freezing
- Pellet, tissues: High pressure freezing
We offer the following EM imaging techniques to visualize your samples:
- Scanning Electron Microscopy (SEM) to visualize the surface topology of samples at room temperature or in cryo-mode.
- Cryo correlative Microscopy : locate the protein of interest in a cell (collaboration with photonic microscopy platform )
After lamella milling:
- cryo-Fluorescence imaging
- Conventional 2D cryo TEM imaging
- Cryo-tomography
Single Particle cryo-Electron Microscopy (SPA cryo-EM)
SPA cryo-EM is widely used for structural analysis of macromolecules or complexes. It aims at the determination of the 3D structure at near atomic resolution of an ideally monodisperse protein or macromolecular complex or virus embedded in vitreous ice. The type of particles that can be imaged range from small proteins (typically above 60 kDa) to large viruses. A typical workflow consists of 3 distinct steps:
- Cryo-EM sample preparation: Specimen vitrification in its native state by blotting and plunging in liquid ethane (Vitrobot IV or Leica GP2).
- Cryo-EM sample optimization: Iterative vitrification & screening to optimize conditions such as ice thickness, particle density, orientation distribution etc.
- Cryo-EM data collection: Acquisition of sufficient number of high-quality movies of the sample embedded in vitreous ice in order to allow structure determination.
The TFS Glacios 200kV cryo-TEM is equipped with a Falcon4i direct electron detector and automated data acquisition software (EPU), which allows collection of a large number of movies of frozen-hydrated macromolecules. It can be used just for grid screening and evaluation of the sample or for data collection and structure determination delivering high resolution 3D reconstructions (typically at the 2.3-4Å range). For challenging cases zero-loss imaging on a 300kV cryo-TEM (e.g., at CM01 or CM02) is recommended, after screening and confirming the sample suitability with a small data collection on the Glacios. . Instruments
Cryo-Electron Tomography (cryo-ET)
Cryo-ET can be used for structural analysis of particles (macromolecules, complexes, viruses etc) and also for thin lamellas of organelles or cells.
Molecular cryo-ET can be used for specimen prepared for single particle analysis, to collect tilt series of the particles embedded in vitreous ice. The reconstructed tomograms can then be analysed in a manner similar to SPA and provide low to medium resolution 3D volumes by sub-tomogram averaging. This technique is developing rapidly but currently has limited use e.g., to generate initial models for SPA, non regular complexes (host-virus complex for example) or for analysing larger objects with repetitive patterns (such as helical assemblies).
Cellular cryo-ET is used for objects such as vesicles, viruses, organelles, bacteria, and even whole cells. Such objects are usually too large to be analysed by other methods. For thicker specimens (organelles, cells etc) preparation of thin lamellas by focused ion beam (FIB) milling at the areas of interest is highly recommended. Tilt series can be collected and then be aligned and reconstructed to generate 3D volumes. Subsequent zero-loss imaging on a 300kV cryo-TEM is advised.
A typical workflow can be divided into the following distinct steps:
- Cryo-EM sample preparation
Specimen vitrification in its native state by blotting and plunging in liquid ethane (Vitrobot IV or Leica GP2) or by high pressure freezing (HPF ICE?) in liquid nitrogen.
- Cryo-EM sample optimization
Iterating vitrification & screening with the aim of optimizing conditions (ice thickness, sample concentration, …).
- Cryo-FIB milling
This step is optional but recommended for thick samples, in order to produce thin lamellae prior to tilt series acquisition.
- Cryo-ET data collection
Acquisition of sufficient number of high-quality tilt series on regions of interest of the frozen-hydrated sample in order to allow reconstruction of 3D volumes. These volumes can also be used for sub-tomogram averaging for higher resolution structure determination of single particles from within the reconstituted tomograms.
The TFS Glacios 200kV cryo-TEM equipped with a Falcon4i direct electron detector and automated data collection software (TOMO or SerialEM) can be used for screening of grids prepared for cryo-ET but also for acquisition of tilt series from vitrified specimen. For higher resolutions and challenging cases, especially for thin lamellae, zero loss imaging on a 300kV cryo-TEM is highly advised (e.g., at CM01 or CM02). Instruments
CryoFIB/SEM milling
This technique is used for lamellae preparation, Volume imaging and cryo-CLEM. The lamellae are thin sections of the sample, produced by milling the excess material; the frozen-hydrated specimen is exposed to a focused ion beam that annihilates part of the sample, while the current of the beam is reduced stepwise at each interval, eventually producing a thin lamella.
This technique is currently under development in our facility following the recent acquisition of an Aquilos 2 dual beam. This microscope is specifically designed for producing thin lamellae (less than 300 nm) of cryo-fixed biological samples that are otherwise too thick for EM (e.g. organelles, cells or protein crystals). Such lamellae are ideal for subsequent imaging by cryo-electron tomography at a cryo-TEM or, in the case of crystals, by MicroED. Instruments
Microcrystal Electron Diffraction (MicroED)
Electron Diffraction, or MicroED, is another modern cryo-EM technique that delivers high-resolution data for a wide range of crystalline samples such as proteins, peptides, as well as organic molecules. Nano-crystals of the molecules of interest are required, which are deposited on an EM grid. The latter can also be blotted and flash frozen by plunging. Subsequently, the small 3D crystals are rotated continuously under the electron beam and at very low doses, while a high-speed detector records the diffraction data as a movie. The crystal structure can then be determined by using standard crystallographic software.
The Tecnai F20 200kV cryo-TEM is equipped with a cryo-tomography holder and an ASI hybrid detector (512 x 512), specifically designed for electron diffraction data. For larger crystals (1 µm or more), the Aquilos 2 can be used for milling thin lamellae (less than 300 nm), well suited for electron diffraction experiments.
Routes of Access
Contact
For any request, please contact ibs-plateforme-em.contact@ibs.fr
Cost
Tariff will be given on request. Rates are eligible for ANR and European funding
Instruct-ERIC access
International access, including sample shipment and travel expenses, can be funded through Instruct-ERIC, see https://instruct-eric.org/centre/instruct-centre-france-2/
Industrial access
Contact us for a quote
Sample Requirement and Handling
Sample requirements vary depending on the technique used and the specific research question being addressed. Detailed guidelines are provided for each technique.
However, all samples must adhere to the following conditions:
- Security Requirements: Samples must comply with biosafety level L1 standards (or L2 if fixed).
- GMO Compliance: Samples must align with the relevant GMO regulations and statements.
After the experiment, samples can either be returned to the customer, if requested, or stored for up to two weeks before being safely discarded.
General guidelines of sample requirements
For Negative Staining
Typically, we ask for ~30 µL of the pure sample (gel filtration, affinity column link to be biophysic PF etc..) at approximately 0.2 mg/mL, with an additional 100 µL of buffer for potential dilution. It is recommended to try to avoid the use of detergent or glycerol in the buffer. The minimum size of molecules that can be observed is 100 kDa.
For Cellular EM
- Morphological analysis: Pure sample
- Immunolabelling: Good antibody, pure sample
- CEMOVIS: Pure and concentrated sample
- Cryo-FIB/SEM: Cells grown on gold grids. Cells under conditions BSL2 are also allowed (due to cryo-fixation).
For cryo-EM samples (SPA or cryo-ET)
We strongly encourage all to verify the sample quality by a combination of: Negative Staining and/or Mass Photometry and any other suitable biophysical technique available to you.
Typically, we ask for ~30 µL of the pure sample at approximately 1-3 mg/mL, with an additional 100 µL of buffer for potential dilution.
It is recommended to try to limit the detergent concentration and avoid use of glycerol in the buffer. The minimum advised size of single molecules to be observed is 60 kDa.
For MicroED
Thin 3D crystals are required (of roughly ~200 nm thickness).
The Team
Guy Schoehn
Scientific director
(+33)(0) 457 428 568
Daphna Fenel
Negative Staining
(+33)(0) 457 428 581
Eleftherios Zarkadas
SPA cryo-EM (& cryo-ET)
(+33)(0) 457 428 581
Christine Moriscot
Cellular EM (RT)
(+33)(0) 457 428 611
Benoit Gallet
Cellular EM (cryo) & FIB/SEM
(+33)(0) 457 428 527
Emma Duscaq
Cellular EM (RT & cryo)
Acknowledgements
Please use the phrase below in any communication including data obtained at the EM platform:
“This work used the platforms of the Grenoble Instruct-ERIC centre (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The IBS-ISBG EM facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the fonds FEDER and the GIS-Infrastructures en Biologie Sante et Agronomie (IBISA).”
You are also strongly encouraged to add: « We thank Guy Schoehn for establishing and managing the IBS-ISBG cryo-electron microscopy platform and for providing access, training and support. » and to acknowledge by name the platform staff who performed the work/training, if they are not included in the author list.
Affiliation for Daphna, Benoit and Guy:
Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
Affiliation for Emma, Christine and Lefteris:
Univ. Grenoble Alpes, CNRS, CEA, EMBL, ISBG, 71 avenue des Martyrs, F-38000 Grenoble, France
Instruments
The IBS/ISBG platform is fully equipped with state-of-the-art microscopes and ancillary instruments to support a wide range of applications. For more information, explore the relevant sections: Techniques and Experiments, Cellular Analysis, 3D Structural Analysis, and Instruments.
Microscopes
1 – Tecnai Spirit T12 (2012)
- Specifications: 120 kV, LaB6, equipped with an Orius 1000 camera (wide field) and a bottom mounted TVIPS F416 camera (4K x 4K).
- Applications: This microscope is used for negative staining and cellular electron microscopy.
Credit: A Delos CEA.
2 – Tecnai F20 (2012)
- Specifications: 200 kV, C-FEG, equipped with a CETA camera and an ASI hybrid detector (1K x 1K). SerialEM software is installed on this microscope. It includes a Gatan 626 cryo-sample holder and a Gatan 614 cryo-tomography holder.
- Applications: This cryo-equipped microscope is used for negative staining, cellular electron microscopy, and cryo-microscopy (single particles as well as CEMOVIS sections). Accessible imaging modes include SPA, tomography, and micro-electron diffraction.
Credit: A Delos CEA.
3 – Talos Glacios (2019)
- Specifications: 200 kV, X-FEG, equipped with a CETA camera, a Gatan K2 Summit direct electron detector, and a Falcon 4i detector. Software such as EPU 3 (multi-grid), TOMO 5, MAPS, Latitude S, and SerialEM is installed on this microscope.
- Applications: This microscope is used to screen grids before transferring them to the Titan Krios. It is also used for data collection in tomography and single-particle analysis (up to 2.2 Å resolution).
4 – Aquilos 2 dual beam cryo-FIB/SEM (2025)
- Specifications: 30 kV, X-FEG. Gallium ionic column. Fluorescent microscope (4 wave length). Possibility of performing lift-out experiment.
- Applications: This instrument is used to mill lamella from massif sample with the aim of performing cryo-electron tomography on them.
Ancillary Instruments
A range of essential auxiliary equipment for sample preparation is also available:
Instruments shared across SPA, tomography, micro-electron diffraction, and cellular electron microscopy techniques:
Instruments specific to cellular electron microscopy:
