FAQs

  • What is cryo Soft X-ray tomography?

    Soft X-ray cryo tomography is an X-ray microscopy technique which allows 3D data to be collected on whole, unstained biological cells or bacterial or potentially other very thin (monolayer) very light carbon containing samples. X-ray microscopy as a technique has been in existence for many years however its application to structural cell biology has only become possible in the past few years due to parallel development of cryogenically cooled soft X-ray microscopes and appropriate sample preparation techniques. Many of the ideas have been taken from the recent developments in cryoEM.

     

    Data are collected using X-rays of ~500eV within the ‘water window’ (the region between the k-absorption edges of carbon at 284eV and oxygen at 543eV where oxygen is virtually transparent to X-rays whereas carbon is highly absorbing). At this energy a natural contrast exists between the vitreous ice surrounding a frozen biological cell and the carbon containing structures making up the cell. Data collected in the water window allow features to be visualized within the cell at a resolution of circa 30-40nm (lipidic entities such as membranes along with high carbon content structures such as mitochondria become clearly visible). All of this assumes a well prepared sample. 

  • Why is it worth trying out this new technique?

    Cryo soft X-ray tomography has the major advantage of being able to collect data through an entire cell (generally under to 9 microns thick) without the need for invasive sample preparation such as heavy metal staining or vitreous sectioning such as might be used for EM. The only sample preparation process is vitrification via plunging in liquid nitrogen cooled liquid ethane. If you are interested in looking at features in the cell which can be damaged in the preparation processes required in your existing techniques (e.g. EM) then the soft X-ray work may well be of use to you.

  • What are suitable samples and how should they be prepared?

    In general the most suitable samples for study with soft X-ray cryo tomography are single layer cells cultured from standard cells lines. This is simply because, at this stage, this is what we have the most experience with though we can also look at other samples such as primary tissue cultures (monolayer), bacteria and yeast.

    The standard sample mount uses a 3mm EM grid coated with a holey carbon film. The cells are grown adherently on the EM grid at low (~50%) confluence prior to being plunge frozen in liquid ethane – in a similar manner to the vitrification of cells prior to cryoEM data collection. Further details and advice is available for those wanting to prepare samples for data collection.

    In the case of bacterial cultures, these can simply be pipetted onto a 3mm grid prior to plunge freezing.

     

    In each case the addition of 250nm gold fiducials to the grids just prior to plunge freezing is strongly recommended in order to aid the alignment of the tilt series which is a key part of data processing. The quality of the tilt series alignment plays a significant part in the resolution of the final tomogram. 

  • Do I need fluorophores in my sample?

    Although fluorescent biomarkers are not necessary for the technique, these can be used to locate suitable cells for imaging and ultimately inform on localisation of biomolecules of interest within the cellular structures that will be visualised.

  • Where can I get grids and fiducials?

    There are several suitable products available on the market such as quantifoil and gold nanoparticles.

     

     

  • How do I process my data?

     We can recomment IMOD which is freely available and we are happy to provide advice and assistance to our users.

  • What can I do at B24 in the run up to X-ray data collection?

    Users can request to access the beamline at different stages of sample preparation, data collection or data analysis.

    Facilities can be made available for:

    • cell maintenance & storage
    • minor biochemical manipulation
    • plunge freezing
    • grid imaging and mapping (widefield and fluorescence)
    • cryo- SIM imaging