Autofluorescence profiles define depot-specific adipocyte phenotypes in perivascular and non-perivascular adipose...

Perivascular adipose tissue (PVAT) is recognized as the functional fourth layer of the vasculature. Unlike non-PVAT depots, PVAT is continuously exposed to haemodynamic forces, potentially altering the morphometry of its adipocytes. While transcriptomics studies have identified diverse adipocyte populations, characterizing these cells remains difficult because their inherent fragility limits standard antibody-based phenotyping. Autofluorescence has emerged as a label-free alternative for characterizing cellular heterogeneity. However, autofluorescence-based characterization of PVAT adipocytes remains unexplored, and it is unknown if these cells exhibit distinct profiles reflecting their specific anatomical location. This study aimed to define the autofluorescence profiles of PVAT and non-PVAT adipocytes. Using Sprague-Dawley rats (n = 6), we assessed intrinsic fluorescence in adipocytes from PVATs of thoracic and abdominal aorta, and from mesenteric arteries alongside phenotype-matched non-PVAT counterparts: interscapular brown, subcutaneous, and retroperitoneal tissues. Using optimized methods to maintain adipocyte integrity, we demonstrate that thoracic and intrascapular exhibit similar autofluorescence profiles. In contrast, PVAT sites mesenteric and abdominal diverge across the yellow, red, and violet channels compared with non-PVAT depots, retroperitoneal and subcutaneous. In conclusion, PVAT and non-PVAT adipocytes exhibit depot-specific spectral patterns, with higher autofluorescence observed in PVAT cells, validating autofluorescence as a marker-independent approach for identifying adipocyte differences.