PORTFOLIO - fjchichon/Guides GitHub Wiki

Techniques and Equipment Portfolio

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1. Negative Staining

Microscope: JEOL 1400 Flash
Description: Technique used for a quick inspection of sample quality before cryopreservation.
Software: SerialEM for automatic dataset acquisition.

Applications:

  • Preliminary evaluation of biological samples.
  • Sample preparation for cryo-electron microscopy studies.
  • Rapid image acquisition for structural analysis.

2. Electron Tomography from Plastic Sections

Microscope: JEOL 1400 Flash
Description: Electron tomography technique applied to plastic sections for three-dimensional sample reconstruction.
Software: SerialEM for automatic dataset acquisition.

Applications:

  • Detailed structural analysis of plastic-embedded samples.
  • Generation of three-dimensional models of biological structures.
  • Study of cellular organization at the ultrastructural level.

3. CryoEM Sample Vitrification

Equipment: Vitrobot Mark IV
Description: Cryo-electron microscopy (CryoEM) sample vitrification system. In the facility, we can test a variety of grid types to analyze the behavior of different particles, including liposomes, viruses, proteins, complexes, and nanoparticles.

Applications:

  • Optimization of grid preparation for CryoEM.
  • Structural studies of various biological and nanomaterial samples.
  • High-quality vitrification for improved imaging and analysis.

4. CryoEM Screening

Microscope: Talos Arctica (200kV)
Description: CryoEM grid screening system. Users can visualize a set of grids by cryo-electron microscopy. A single 24-hour session allows screening of up to 12 grids to verify sample quality and proper grid positioning.

Applications:

  • Rapid evaluation of sample integrity and ice thickness.
  • Verification of grid quality before high-resolution data collection.
  • Optimization of sample preparation for structural studies.

5. CryoEM Screening and Dataset Acquisition

Microscope: Talos Arctica (200kV) with Falcon 4i
Description: CryoEM system for grid screening and dataset acquisition. Users can visualize a set of grids via cryo-electron microscopy. A single 24-hour session allows screening of up to 8 grids, with the option to set up a Multigrid Acquisition Session using two or three grids. A standard overnight (O/N) acquisition can produce between 6000 and 7000 movies. Using two grids, this results in two datasets of approximately 3000 to 4000 movies per grid.

Applications:

  • Screening and dataset acquisition for CryoEM structural studies.
  • Optimization of data collection strategies for high-resolution imaging.
  • Efficient use of grid samples to maximize data output.

6. CryoEM Dataset Acquisition

Microscope: Talos Arctica (200kV) with Falcon 4i
Description: High-resolution CryoEM dataset acquisition system. Users can screen up to 4 grids and set up a high-resolution dataset acquisition. A typical session can produce between 6000 and 9000 movies. Our results with apoferritin and the Talos system currently achieve a resolution below 2 Å.

Applications:

  • High-resolution structural studies using CryoEM.
  • Data acquisition for atomic-level resolution imaging.
  • Benchmarking and optimizing sample preparation for ultra-high-resolution reconstructions.

7. CryoEM Data Processing and Analysis

Software: ThermoFisher cryoFlow & Scipion
Description: Integrated CryoEM data processing workflow. The facility provides users with the capability to align movies using ThermoFisher cryoFlow. If the sample is suited for automatic picking, 2D and 3D processing can be programmed for fast screening of structures. Standard electron microscopy image processing is conducted using Scipion.

Applications:

  • Automated movie alignment for high-throughput processing.
  • Fast 2D and 3D structure screening.
  • Comprehensive image processing for CryoEM datasets.

8. Cryo-Electron Tomography

Microscope: Talos Arctica (200kV) with Falcon 4i
Software: ThermoFisher Tomography 5
Description: Cryo-electron tomography acquisition system. The software enables fast and accurate tilt series acquisition, allowing the collection of approximately 100 series in a 24-hour session.

Applications:

  • High-throughput cryo-electron tomography for structural biology.
  • Three-dimensional reconstruction of complex biological samples.
  • Efficient acquisition of tilt series for in-depth sample analysis.

9. Automatic Reconstruction of Tilt-Series

Software: ThermoFisher cryoFlow & Scipion Tomography
Description: Automated workflow for tilt-series alignment and reconstruction. Both ThermoFisher cryoFlow and Scipion Tomography allow users to align and reconstruct data immediately, enabling fast analysis without requiring extensive knowledge of tomography reconstruction techniques. This minimizes processing time and maximizes efficiency.

Applications:

  • Automated reconstruction of tilt-series for cryo-electron tomography.
  • Immediate data analysis without the need for extensive user intervention.
  • Streamlined tomography workflows for fast structural interpretation.

10. Cryo-Light Microscopy

Equipment: Linkam Stage, Widefield and Confocal Microscopes (Axio S1 and LSM 900 AiryScan)
Description: Cryo-light microscopy system for imaging vitrified grids under cryogenic conditions. Cells grown on gold EM grids are typically screened using widefield microscopy, followed by confocal imaging using the LSM 900. The AiryScan system allows for enhanced resolution despite the high working distance.

Applications:

  • Imaging of vitrified grids under cryogenic conditions.
  • Rapid screening of cells grown on EM grids.
  • High-resolution multicolor confocal imaging with AiryScan technology.

11. Cell and Tissue Vitrification

Equipment: High-Pressure Freezing Device (Leica ICE)
Description: Monolayer cell lines can grow over gold EM grids and be vitrified by plunge freezing for EM visualization. Suspension cell lines, viruses, or large nanoparticles can also be deposited and fixed on EM grids. However, tissues and thicker samples require a different approach. To address this, we use a high-pressure freezing device (Leica ICE), which ensures optimal vitrification of thick samples.

Applications:

  • Vitrification of monolayer cell cultures for cryoEM studies.
  • Preservation of suspension cell lines, viruses, and nanoparticles.
  • High-pressure freezing of thick biological samples for structural analysis.

12. Volume Imaging with FIB-SEM (Plastic Blocks)

Microscope: ZEISS Crossbeam 550 with Leica Stage
Description: Volume imaging of plastic blocks (Epon and Lowicryl) using focused ion beam-scanning electron microscopy (FIB-SEM). Provides cellular resolution (~10 nm). In a single day, volumes of approximately 10 µm wide and several µm deep can be acquired.

Applications:

  • High-resolution volumetric imaging of resin-embedded biological samples.
  • 3D reconstruction of subcellular structures.
  • Detailed cellular architecture analysis.

13. Volume Imaging of Vitrified Tissue

Microscope: ZEISS Crossbeam 550 with Leica Stage
Description: Volume imaging of vitrified tissue (as prepared in Section 11) using FIB-SEM. Achieves similar resolution (~10 nm), though with slightly lower contrast and slower data acquisition compared to plastic blocks.

Applications:

  • 3D imaging of vitrified biological tissues.
  • Structural analysis of cryo-preserved thick samples.
  • Complementary technique to plastic block imaging for cryo studies.

14. Cryo-Lamella Generation

Microscope: ZEISS Crossbeam 550 with Leica Stage
Description: Lamella generation from cryo-fixed samples on grids, using the same FIB-SEM system described in Sections 12 and 13. Samples are vitrified either by plunge freezing or high-pressure freezing using the waffle method.

Applications:

  • Preparation of thin lamellae for cryo-electron tomography or cryoEM.
  • Site-specific thinning of vitrified biological material.
  • Enabling high-resolution imaging of internal cellular structures.

15. Cryo-Lift-Out

Microscope: ZEISS Crossbeam 550 with Leica Stage and Kleindiek Microgripper
Description: Using the same FIB-SEM system, equipped with a Kleindiek micromanipulator, it is possible to extract a small lamella from a vitrified or plastic block and transfer it to a dedicated grid for cryo-electron tomography (as described in Section 8).

Applications:

  • Cryo-lift-out from thick samples or blocks.
  • Transfer of lamellae for tomography analysis.
  • Targeted sample preparation for 3D electron microscopy.

16. Electron Diffraction / MicroED

Microscope: Talos Arctica
Description: This technique captures electron diffraction patterns from crystals, compounds, or proteins to determine their atomic structure. Particularly useful for samples that do not form crystals large enough for traditional X-ray diffraction.

Applications:

  • Atomic structure determination of small crystals.
  • Structural studies of compounds not suitable for X-ray diffraction.
  • Complementary technique to X-ray crystallography.

17. Correlative Approach

Equipment: Combination of Cryo-Light Microscopy (Section 10), FIB-SEM, and TEM
Description: Correlative microscopy allows fluorescently labeled events to be localized and analyzed using a combination of cryo-light microscopy, FIB-SEM, and TEM techniques. This approach enables a detailed understanding of spatial and molecular organization within complex biological samples.

Applications:

  • Multimodal imaging of labeled structures.
  • Integration of fluorescence, electron, and volume data.
  • Comprehensive structural and molecular analysis in situ.