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Communication Dans Un Congrès Année : 2020

Leveraging Molecular and Cellular Fiducial Markers for Correlative Bio-Imaging

Résumé

Beamline B24 is the biological correlative cryo-imaging beamline at the UK synchrotron Diamond Light Source, delivering 3D soft X-ray imaging and structured illumination fluorescence imaging of cryopreserved biological materials such as cells and tissues to nanometer resolution. Our unique, semi-automated and user-friendly correlative microscopy workflow offers unambiguous multidimensional localisation of biomolecules within the cellular suprastructure and is available to the scientific community via a peer-reviewed application process. Cryo Soft X-ray Tomography (cryo-SXT) 3D data allows the identification and quantification of cellular ultrastructure and subcellular organelles. It is used to study the structural effects of biological, chemical and mechanical cues on cell populations and provides wide field cellular context for data from other imaging modalities such as fluorescence microscopy. At B24, fluorescence localisation data is provided by the structured illumination super resolution fluorescence microscopy method using a bespoke cryo-structured illumination microscope (cryo-SIM). The particular attraction of this correlative imaging system is that samples that are due to be used for X-ray imaging can be first imaged on the cryoSIM to gather 3D fluorescence information at high resolution and identify areas of interest before being taken for cryo-SXT, thereby allowing for the accumulation of easily correlatable and unambiguous localisation data. Effectively, the same cryogenically preserved near-native cell population can now be imaged at nanometer resolution through a variety of imaging methods within and beyond the diffraction limit of available light sources. The resulting data is not compromised by chemical fixation, mechanical milling or slicing, additional staining or sample-to-sample variations and therefore allows the unambiguous interpretation of imaging data across modalities to nanometer resolution. Recently, as part of a project to ensure accurate information capture and precise correlation across state-of-the-art imaging modalities, we have carried out a comparative study using different commercial nanoparticles and bio-trackers for correlation of X-ray and fluorescence images. We have analysed size, distribution and contrasting properties of a range of potential biomarkers and their relative contribution to correlative analysis of real imaging data. We present here a novel biological fiducialisation system, which uses a combination of nanoparticles and biomarkers to ensure minimal disruption of physiological cell structures while delivering precise correlation of data through an intuitive and user-friendly in silico workflow.
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Dates et versions

hal-04219534 , version 1 (27-09-2023)

Identifiants

  • HAL Id : hal-04219534 , version 1

Citer

Chidinma Okolo, Ilias Kounatidis, Thomas Fish, Perrine Paul-Gilloteaux, Ian Dobbie, et al.. Leveraging Molecular and Cellular Fiducial Markers for Correlative Bio-Imaging. Quantitative BioImaging, Jan 2020, Oxford, United Kingdom. ⟨hal-04219534⟩
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