Correlative soft-X ray cryo-tomography and super resolution fluorescence microscopy imaging for the life sciences at Diamond Light Source beamline B24.

Maria is the acting Principle Beamline Scientist for beamline B24. Previously she was a group leader at the Structural biology laboratory at the Nuffield department of Medicine of the University of Oxford. Maria undertook her graduate studies at YSBL, York, and was awarded her PhD in 2002.

B24 is the full field X-ray tomography beamline at Diamond currently delivering X-ray absorption contrast imaging of biological material (cells and tissue sections) to a resolution of 40nm. The resulting cryo Soft X-ray Tomography (cryoSXT) 3D data allows the unambiguous delineation of cellular ultrastructure and is employed in the interpretation of the effects of biological chemical and mechanical cues depending on the subject matter. The B24 soft X-ray microscope is currently fully operational and available to the wider user community.
CryoSXT also provides context for further investigation on the molecular level. The latter is gained via super resolution fluorescence microscopy methods that provide invaluable information as to the molecular localisation of key parameters relevant to the system under study within the context of cellular maps defined via cryoSXT. To that effect, at B24, a bespoke cryo fluorescence super resolution microscope has been developed offering both cryo-Structured Illumination Microscopy (cryoSIM) and dSTORM. The particular attraction of the system is that samples due to be used for X-ray imaging can be processed there first to generate 3D fluorescence information at high resolution on identified areas of interest before taken to the transmission X-ray microscope. This allows the accumulation of directly correlated localisation data (the same sample is imaged through a variety of methods) eliminating sample to sample variations and allowing the unambiguous interpretation of data across modalities. The B24 cryoSIM capacity is at present fully implemented and is available to the user community on a commissioning basis. dSTORM is implemented optically but has not been tested with samples yet. Software is being developed to handle data processing and correlation across imaging modes.
The beamline workflow will be presented with examples of recent data collected along with highlights and pitfalls of the correlative scheme employed on site.