Abstract Background Analysis of myocardial biopsies using conventional two-dimensional (2D) histological techniques is limited in the detection of important three-dimensional (3D) structural features, such as disarray, due to sectioning and artefacts from dehydration or chemical processing. More advanced techniques such as light-sheet microscopy, micro-computed tomography or synchrotron radiation-based X-ray phase contrast imaging can provide non-destructive, 3D histological information at high resolution for formalin-fixed and/or paraffin-embedded (FFPE) biopsies. However, formalin fixation induces crosslinking of proteins which can impact quality and their subsequent analysis, for example, fragmentation of nucleic acids. A method for imaging 3D structure of myocardial biopsies in near-native state is needed which can then be followed by downstream multi-omic applications. Purpose To develop a technique called cryogenic X-ray phase contrast imaging (Cryo-X-PCI) to image snap-frozen myocardial biopsies at controlled low temperatures, followed by multi-omic analysis starting with analysis of DNA and RNA. Methods Myocardial biopsies (n=46) were obtained from C57BL/6 mice and snap-frozen in liquid nitrogen fresh or after 4% paraformaldehyde (PFA);10% formalin fixation. Pilot human aortic stenosis biopsies were also included (n=3, fresh). X-PCI was performed at -70°C using a cryojet, 20 keV beam energy and 0.65 µm detector pixel size (Figure 1). Datasets were reconstructed using Gridrec algorithm and Paganin phase retrieval. Morphologic analysis (myocardial disarray index, fractional anisotropy, helical angle, and intrusion angle) of biopsies was performed using a structure tensor-based approach via in-house MATLAB script. After imaging, DNA and RNA were extracted from murine biopsies followed by assessment of quantity and quality. Results Increased intercellular space between aggregates of myocytes was observed in all samples. This is consistent with reported findings in skeletal muscle using cryogenic conditions. Nonetheless, morphologic analysis of the biopsies proved possible (Figure 2). Optimal recovery of DNA and RNA was obtained from fresh frozen samples as compared to 4% PFA-fixed or 10% formalin-fixed samples. Conclusions This study demonstrates Cryo-X-PCI as a potential tool for analysing myocardial biopsies of heart muscle disease including morphologic analyses. Levels of DNA and RNA were not significantly affected indicating that downstream genomics, proteomics and transcriptomics analyses should be feasible.Setup for Cryo-X-PCI of frozen biopsiesMorphologic analysis of a biopsy
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