Service / Technology Instance

Based at the premises of the Centro Nacional de Biotecnología (CNB), the CSIC cryoelectron microscopy (CryoEM) facility is a state-of-the-art core service that offers both sample preparation and CryoEM image collection of unstained biological material.

The facility hosts two cryoelectron microscopes: A 300 kV JEOL CryoARM equipped with an autoloader, a Gatan K3 direct electron detector and an Omega energy filter, and a 200 kV FEI TALOS Arctica, equipped with an autoloader and a Falcon 4i direct electron detector. Both are suited for the collection of large amounts of high-resolution data that can be used for high-resolution studies using single-particle methodology, or for the reconstruction of pleiotropic structures such as cells and subcellular structures using cryoelectron tomography (cryoET). Additionally, the facility hosts a standard 120 kV electron microscope JEOL JEM1400 for sample screening.

Specimen vitrification is also available at the service, with two devices suited for vitrification of small specimens such as proteins and macromolecular complexes (a FEI Vitrobot and a Leica EM CPC), and a third device suited for the fast-freezing of cellular samples, a Leica EM GP2 high-pressure freezer. This facility is unique in Spain as it also offers the newly developed cryocorrelative microscopy technique, which allows the analysis of the same biological specimen by cryooptical microscopy (Zeiss LS900 AiryScan cryoconfocal microscope) and cryoelectron microscopy (both cryoelectron microscopes described above). The use of a Zeiss CrossBeam 550 cryo-FIB-SEM microscope will increase the cryocorrelative microscopy capabilities of the facility to direct visualization of cells for tissue-cell resolution or for preparation of thin lamellas in cells for molecular resolution.

Vitrified cellular or tissue samples on grids or planchettes will be analysed by cryoconfocal microscopy (Zeiss LS900 AiryScan) to gain functional and location information using a Linkam stage. Analysed samples will be transferred to a Zeiss CrossBeam550 microscope together with the fluorescence information collected to perform cryo-volume imaging (organelle resolution level) of the selected area. Samples could be provided by the user or from a previous project in our facility of cryoCLEMsample characterisation.

It is extremely important to contact the facility (cryoemcsic_facility@cnb.csic.es) before submitting the proposal, to address the technical viability of your experiment and discuss the details on how they should be done.

The facility will provide support and expertise to researchers in the optimisation of cellular or tissue sample preparation for cryoCLEM workflow including plunge freezing (FEI Vitrobot Mark IV, Leica EM CPC and Leica GP2 ) and high pressure freezing (Leica ICE). Once vitrified, sample screening and selection will be performed by cryofluorescence microscopy using a Linkam stage coupled to either a widefield or confocal microscope (Zeiss LS900 AiryScan).

It is extremely important to contact the facility (cryoemcsic_facility@cnb.csic.es) before submitting the proposal, to discuss the technical feasibility of your experiment and discuss the details on how it will be carried out.

a) Single particle High-end data acquisition will be performed with a 300 kV JEOL CryoARM equipped with an autoloader, a Gatan K3 direct electron detector and an Omega energy filter. High-quality cryogrids could either be provided by the user or come from a successful sample characterisation project.

b) Cryo-EM tomography Data acquisition will be performed with a 300 kV JEOL CryoARM equipped with a Gatan K3 direct electron detector and an Omega energy filter. Cryo-grids could be provided by the user or from a previous project in our facility of sample characterization (it is advisable to contact the Instruct Image Processing Centre for support in the data processing).

Micro-Electron Diffraction data are acquired in a Talos Arctica 200 kV microscope equipped with a scintillation-based Ceta-D camera optimised for low dose data acquisition, ideal for projects involving dose-sensitive samples such as proteins, peptides, pharmaceutical compounds and organic materials.

Basic workflow for sample handling and data acquisition:

• Samples are placed on grids, and vitrified if in solution, under different conditions depending on the nature of the sample.
• Procedures for powdered compounds: Samples are crushed and pulverised by mechanical methods and applied to microscope slides.
• Methods for peptide and protein crystals: Crystals are collected from the mother liquor solution and applied to grids for vitrification under various blotting conditions.
• General data collection procedure: Grid quality control: ice contamination and thickness in the case of vitrified samples, crystal distribution on the grid, blotting quality. Crystal search is performed in semi-automated mode according to an in-house developed workflow based on SerialEM software (Mastronarde, D.N. 2003. SerialEM: A Program for Automated Tilt Series Acquisition on Tecnai Microscopes Using prediction of Specimen Position Prediction. Microscopy and Microanalysis Vol. 9, Suppl. 2, 1182CD.), a preliminary diffraction quality check is performed, and if a quality diffraction pattern is obtained, data are acquired for preliminary processing until the data are complete.

Mandatory information:

• Ensure that it is crystalline material
• Safety declaration for all the samples certified by user’s home Institution, especially if volatile reagents are used.
• Sufficient sample to be processed by humans.
• A brief report specifying:
  • Sample name or code. An inhouse code will be assigned and supplied to the user.
  • Type of sample: pharma compound, organic compound, peptide, protein.
  • Is it a pure sample or mix?
  • Chemical formula/composition in atoms or amino acid sequence. Structure diagram if available.
  • Concerning protein crystals, enclose crystallisation buffers and information about possible cryo-conditions.

If previous diffraction experiments have been performed, a very short description of the results: problems, unit cell, space group, crystal shape and size.

The facility will provide support and expertise to researchers with samples in the first stages of characterisation. Samples will be analysed by negative staining in search of the best conditions. Once found, samples will be subjected to different vitrification conditions in search of the best parameters, which will be tested in a cryomicroscope Talos Arctica 200 kV equipped with a Falcon 4i electron direct detector. The best grids will be used to acquire some data for image processing, to check the real quality of the sample (it is advisable to contact the Instruct Image Processing Centre for support in the data processing).

1. Negative stain to check if the sample has some problems during the shipping.
2. Samples will be subjected to different vitrification conditions in search of the best parameters.
3. Grids will be tested in a cryomicroscope Talos Arctica 200 kV equipped with a Falcon 4i electron direct detector.
4. Step 2 and 3 will be repeated up to the end of the access if necessary.
5. If the quality of the sample is good, some data will be acquired for 2D image processing for a deeper understanding of the sample performance (it is advisable to contact the Instruct Image Processing Centre for support in the data processing).