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