MICROSCOPY & IMAGING
Novel approach to investigate the
Beamline scientists, Maria Harkiolaki and Ilias Kounatidis, review a new microscopy platform that specialises in imaging the fine details of cellular ultrastructure and chemical localisation in 3D
A
t the UK’s synchrotron, Diamond Light Source, a novel combination of imaging on beamline B24 is enabling high-resolution 3D imaging of the cellular universe. It uses a partnership of two cutting-edge methods: soft X-ray tomography (SXT) that delivers 3D scans of structures inside and across cells to a resolution of 25nm; and structured illumination microscopy (SIM), which pins chemical information on those scans through fluorescence and super resolution imaging. Tis combined correlative light microscopy and X-ray tomography (CLXT) platform can deliver content beyond the reach either method can deliver alone. Within this scheme, correlated 3D imaging data is acquired through an established workflow that sees a sample go from one microscope to the other
CELLULAR WORLD
without any mechanical or chemical processing. Tis, in turn, ensures that the 3D cellular data captured in one microscope absolutely corresponds to the 3D data captured on the next, making the association of features of interest unambiguous and, therefore, immediately credible as it circumvents the need to infer similarities.
KEY TO SUCCESS IS SAMPLE CYROPRESERVATION Beamline B24 is a Phase III beamline and laboratory that set out to deliver much needed 3D imaging across resolution scales and capture data deep inside cells at near physiological conditions. However, this could only have been achieved through sample cryopreservation. So the journey a sample takes through imaging at beamline B24 begins when it is rapidly
The B24 platform allowed the transition of information from cytoskeleton fluorescence, to X-ray projections of the cytoplasm, to the full correlation of both
plunge frozen in cryogenic liquid. Tis immobilisation step is required to preserve delicate cellular structures in a state representative of the point in time they were harvested. Cryogenic temperatures complicate
microscope design and sample handling but allow researchers to use intense X-ray and laser light regimes to see features in great detail to a few tens of nanometres even if they reside deep inside cells. For reference, a single Covid-19 particle is just over 100nm in diameter. Tese features can be delineated in X-ray projections and, if they are tagged with fluorophores that relate information on their current state, they can be chemically characterised. Te development of this correlative platform has only recently been fully documented in a publication[1]
, using
as a proof-of-concept this experimental approach to study viral infection processes in human bone osteosarcoma cells. In it, the B24 CLXT shed light on reovirus behaviour during the early stages of infection and provided data on the
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