Cryo-X-Ray Phase Contrast Imaging Enables Combined 3D Structural Quantification and Nucleic Acid Analysis of Myocardial Biopsies

Abstract

Snap-frozen biopsies serve as a valuable clinical resource of archival material for disease research, as they enable a comprehensive array of downstream analyses to be performed, including extraction and sequencing of nucleic acids. Obtaining three-dimensional (3D) structural information before multi-omics is more challenging but can potentially allow for better characterization of tissues and targeting of clinically relevant cells. Conventional histological techniques are limited in this regard due to their destructive nature and the reconstruction artifacts produced by sectioning, dehydration, and chemical processing. These limitations are particularly notable in soft tissues such as the heart. In this study, the feasibility of using synchrotron-based cryo-X-ray phase contrast imaging (cryo-X-PCI) of snap-frozen myocardial biopsies is assessed and 3D structure tensor analysis of aggregated myocytes, followed by nucleic acid (DNA and RNA) extraction and analysis. It is shown that optimal sample preparation is the key driver for successful structural and nucleic acid preservation which is unaffected by the process of cryo-X-PCI. It is proposed that cryo-X-PCI has clinical value for 3D tissue analysis of cardiac and potentially non-cardiac soft tissue biopsies before nucleic acid investigation.

Keywords: biopsies; cryo‐X‐ray phase contrast imaging; genomics; nucleic acid analysis; transcriptomics.

Figure 1

Schematic of synchrotron‐based cryo‐X‐PCI setup and workflow for combined 3D, non‐destructive myocardial morphologic assessment, and nucleic acid analysis.

Figure 2

Myomapping and DNA integrity in representative fresh frozen, 4% paraformaldehyde‐fixed (4% PFA) and 10% formaldehyde‐fixed (10% F) mouse myocardial biopsies after cryo‐X‐PCI. An expected gradual change in HA could be observed, from positive HA in the endocardium (endo) to negative HA in the epicardium (epi).

Figure 3

DNA quality control TapeStation gels with DNA Integrity Number (DIN) values of gDNA extracted from representative mouse myocardial biopsies after synchrotron‐based cryo‐X‐PCI. Key: 4% PFA = 4% PFA‐fixed samples; 10% F = 10% formaldehyde‐fixed samples. Control samples represent those that were collected in RNAlater solution then snap frozen in liquid nitrogen and did not have cryo‐X‐PCI. DIN values appropriate for downstream analysis are shown in green. DIN values unsuitable for downstream analysis are shown as red including those that could not be calculated due to very low concentrations of DNA.