tAF-MUSICAL: Autofluorescence Super-resolution Microscopy for Molecular Histopathology of Matrix Proteins in Fibrotic Diseases

Abstract Fibrosis is an extracellular matrix disease affecting several vital organs' functions and can lead to life-threatening pathologies like cancer. The standard diagnostic protocol involves an immuno-histochemical examination of the fibrosis-linked protein's distribution in the tissues. Of specific interest are the primarily affected dense matrix-proteins like collagen. But labeling such dense proteins is prone to subjectivity. Besides distribution, the collagen nano-fibril characteristics, usually revealed by ultra-structural imaging, also have diagnostic relevance. Super-resolution microscopy can aid high-resolution clinical decisions by imaging protein nano-structures but is challenging for dense proteins. Here we exploit the natural fluctuations of tissue autofluorescence (tAF) signal from proteins in dense tissue matrix for super-resolving them. We achieved super-resolution over a clinically relevant large area using a simple, low-cost, low numerical aperture (NA) optical microscope and a recent fluctuation-based nanoscopy technique MUSICAL. We could quantify protein distribution and nano-fibril dimensions as low as 43 nm in human oral mucosa with pre-cancer fibrosis and mouse skin samples to assess disease progression. Thus tAF-MUSICAL enables early, label-free, and high-resolution diagnosis of matrix-associated diseases like fibrosis.