-> Ultrastructural details of surface features
-> Secondary electron, back-scattered electron detectors
-> Energy Dispersive X-Ray Spectroscopy
-> Variable pressure mode (Hydrated biological samples can be seen without much sample processing)

-> Ultrastructural details of thin samples
-> Bright Field Imaging
-> Tomogram generation

-> 3-D imaging of biological samples at micrometric resolution
-> Non-invasive, non-destructive method for tomogram regeneration of objects several mm in dimension
-> Routinely used for mineralized samples like teeth and bone; other tissues like muscles etc.

Equipment available: Titan Krios G31, a 300 kV electron microscope

Accessory instruments
-> Freezing grids: An important step in the cryo-EM is specimen preparation, which involves depositing a protein solution on grid, blotting excess fluid and rapid freezing. The pre-treatment of grid that is to make it hydrophilic or hydrophobic is done with a glow discharge apparatus. This allows the protein solution to spread onto the grids. The grids can then be used for freezing with a Vitrobot, which has a controlled chamber for maintaining humidity and temperature.

-> Quorum Glocube
-> Quorum Carbon Evaporator
-> Vitrobot mark IV
-> Edwards Benchtop Carbon Evaporator, BT150

In electron cryo microscopy, macromolecules in solution are frozen rapidly and then imaged with electrons. The images, which are projections of the molecule of interest are then averaged and reconstructed to obtain a high-resolution map. As the molecules are frozen from a solution the possibility to get multiple conformational or structural states is an added advantage with Cryo-EM.

From the theoretical estimate, it is clear that averaging few thousand asymmetric units is sufficient to obtain 2.5-3.5 Å map and this is true for various molecular sizes of protein. Although, molecules as small as 14 kDa can be observed by Cryo-EM these do not have enough information for high-resolution reconstruction with current technology. Protein molecules such as haemoglobin (64 kDa) has been determined to high-resolution by Cryo-EM and the lower limit of the size will keep decreasing.

Any research group, institutes and companies, who would like to use the Electron Cryo Microscopy facility to collect EM images should submit a proposal, which will undergo peer-review process. After the proposal has been evaluated by reviewers, time will be allocated typically in the beginning of the week.

We will be happy to provide advice on data processing but currently we will not be able to give access for data processing.
A wide variety of specimen can be imaged with EM at cryo temperatures. It is important to remember that the quality of the specimen (in case of single particle cryo-EM, biochemistry of the protein) and the grid preparation largely determines the outcome of the data collection.

A biochemically well characterised protein solution will in most cases result in very good grids and images. Thus, the data collection and processing become easier. Heterogeneity per se is not a big issue as classification during processing can be used to sort through different populations but if this can be addressed biochemically then it saves time. Our advice is to be realistic and very often one finds that a sample that gives a good gel filtration profile or a single band in gel might look completely different on a micrograph. Please do not blindly follow the protocols described in publications but try to explore conditions with the protein of your choice.
 

 
Useful tips:
1) The amount of protein required for making an EM grid will depend on many factors (including the size of the protein, if a carbon or graphene layer is used). For a rough estimation of how much protein is required to image in ice, please refer to the table in the review – Vinothkumar & Henderson, Quarterly Review of Biophysics, 2016.

2) High-resolution structures of small proteins such as Haemoglobin (64 kDa) has been determined by single particle EM and this lower limit is likely to reduce as we understand more about imaging and technology develops. While such molecular sizes are doable, such projects take a lot of effort and when possible, crystallization and X-ray Crystallography should be carried out in parallel.

3) More images or particles doesn’t guarantee increase in resolution. Often one would find that the resolution is limited due to the nature of protein in particular as many are dynamic. As an initial step it is useful to collect a small to medium size data set (no more than 700 images or 30-40,000 particles), check if the refinement and reconstruction goes well. Using the B-factor estimated from the data (see Rosenthal and Henderson 2003), it is possible to estimate how many more particles you will need to attain the resolution of your interest. We highly recommend this before collecting large data sets, which will require a number of days of computing yet may not result in higher resolution.