Techniques and Services

Scanning Electron Microscopy (SEM)

A focussed electron beam scans the surface of the sample and generates a rendering of the surface.

How EMF can help: EMF houses a Tescan Vega3 LMU Scanning Electron Microscope with Oxford X-Max 50 EDS Detector for SEM imaging. The facility provides basic reagents and training for sample preparation and imaging.

Transmission Electron Microscopy (TEM)

An electron beam passes through the sample and provides structural information about both the sample surface and interior. TEM can  be performed at room temperature or at cryogenic temperature (cryoEM). Room temperature EM experiments are typically easier to set up compared to cryoEM experiments, but cryoEM provides higher resolution, mostly artifact-free images of biological macromolecules in their frozen hydrated state. Routine screening is performed at room temperature, before moving to cryoEM for higher resolution information, if needed.

Room temperature TEM

Negative stain EM

A commonly used room temperature TEM method is negative stain EM. Here the sample is coated with a layer of heavy metal salts,such as uranyl acetate, and this coating generates the contrast required for visualisation. One can visualise samples at a range of different sizes using this method, ranging from proteins as small as ~ 25 kD to large viruses and phages with molecular weights of several MD. This method is suitable for checking the purity and homogeneity of the sample and resolution is limited to ~20 Å. Typical sample requirement ranges from 100-200 nM and ~4 µl sample is needed per experiment.

How EMF can help: EMF houses a brand new Jeol 120i EM for imaging negative stain samples. Using this microscope, 4-8 samples could be imaged in a few hours. Additionally, the facility provides basic reagents and training for sample preparation and imaging.

Serial sectioning

Another room temperature approach involves embedding the sample in resin blocks and then performing serial sectioning of the resin-embedded sample. The sections are then visualised under an electron microscope. This method is suitable for visualising cellular ultrastructure. While this is a popular approach the user needs to be careful about artifacts arising from resin embedding and the sectioning process. 

How EMF can help: The 120i is an excellent microscope for visualising these sections. EMF provides access to the microscope and the instrumentation needed for sectioning, along with training for operating the 120i. At present we unfortunately cannot provide training for sample preparation.

CryoEM

SPA

This method is excellent for looking at high-resolution (4 Å and better) 3D reconstruction of macromolecules ranging from individual proteins (as small as 50 kDa) to several MDa ribosomes, viruses and bacteriophages. This method requires ~4 µl of purified samples per experiment with the required concentration ranging from 200 mM to ~40 µM. Typical sample concentration ranges from 1 to 3 µM. We would recommend starting with at least 20µl sample. The sample is applied on a EM grid and after blotting away the excess buffer, the grid is flash frozen in liquid ethane. This prevents the formation of damaging crystalline ice and instead leads to the formation of glass-like amorphous ice. This way the specimen is preserved in its near-native state. The frozen sample is then imaged in a cryo-electron microscope. 

SPA requires extensive image processing. The raw data that comes out of the microscope are low contrast images of the sample. Using multi-step image processing, the signal from tens of thousands of such images are averaged and “stitched” in 3D space to create a high-resolution 3D reconstruction of the specimen.

How EMF can help: EMF houses a state-of-the art FEI Tecnai Arctica cryo-electron microscope with a very bright and coherent FEG electron source, an ultra-stable stage, automated sample loading and unloading capacity. This microscope is equipped with an ultra-sensitive FEI FalconIII camera (FalconIII is a direct electron detector and can literally count the individual electrons hitting the camera chip). The microscope can be operated by EPU software for automated data collection. We have benchmarked this microscope by solving the structure of horse spleen apoferritin to 2.6Å resolution. EMF also houses two flash freezing devices, Leica cryo GP and FEI Vitrobot MachIII along with associated grid pre-preparation instruments.

EMF provides training for safe and effective use of the cryo-electron microscope and all the cryo-grid preparation instruments. While the time requirement for SPA experiments is heavily specimen-dependent, after training, initial grid optimization and data collection can be performed in as short a time as 6-8 weeks.

CryoET

This approach is suitable for samples for which we do not have multiple copies that can be averaged. In this method the specimen stage is systematically titled to provide the angular coverage required for 3D rendering. This method is an excellent choice for visualising cellular ultrastructure. If the sample is too thick for EM (e.g. eukaryotic cell) then the user might need to “burn off” most of the sample and prepare a thin lamella (100 to 300 mm thick) spanning the region of interest. This lamella can then be visualised under cryoEM in great detail. If there are repeating substructures within a tomogram then those can be averaged to form high resolution 3D reconstruction of that substructure.

How EMF can help: The Tecnai Arctica is capable of collecting tomograms and is equipped with automated tomogram collection software. In addition to the flash freezing devices mentioned above, EMF houses a Lecia high pressure freezer. This device is suitable for freezing relatively thick specimens without forming crystalline ice. Training is provided for the safe and effective use of all these instruments.

EMF currently does not have the capacity to generate lamella. If your project requires lamella generation then you may want to use the national EM facility at eBIC. EMF can provide instrumentation access and training needed to optimally flash freeze the sample on a grid. This is an essential step for using the lamella generation pipeline at eBIC.

Correlative light and electron microscopy (CLEM)

This approach sits at the interface of fluorescence microscopy and EM. Using this method a fluorescent signal detected using a light microscope can be correlated with the corresponding electron micrograph. The primary requirement is for the specimen to have some traceable fluorescent signal (e.g. a fluorescent tag like GFP). The sample is first imaged using a CLEM microscope and a map of the specimen is prepared with a detailed annotation of where the fluorescent signal is localised. The same sample is then imaged using an electron microscope and then the EM micrograph is correlated with the fluorescent signal map. This approach can provide rich information about the surrounding area where the fluorescence is localised. CLEM can be performed both at room temperature and under cryo conditions. For room temperature CLEM the sample is typically resin-embedded while in cryo-CLEM the sample is flash frozen and might require lamella generation.

How EMF can help: EMF houses a Leica CLEM microscope with the corresponding mapping software for the fluorescent signal. On the EM side, room temperature specimens can be visualised using the 120i and cryo-specimen can be visualised using the cryo-electron microscope. If the sample is thick then lamella formation is required prior to visualisation.

While EMF offers access to all the instruments, currently no training is provided for CLEM approaches.