Label-Free Whole Slide Virtual Multi-Staining Using Dual-Excitation Photon Absorption Remote Sensing Microscopy

Abstract

Histochemical staining is essential for visualizing tissue architecture and cellular morphology but is destructive and limited by the availability of tissue for multiple stains. Virtual staining with label-free microscopy offers a non-destructive alternative, enabling multiple stains to be generated from the same section while reducing stain variability and preserving tissue for downstream assays. Here, a new dual-excitation Photon Absorption Remote Sensing (PARS) system is presented, representing the first application of long-wave ultraviolet A (UVA) 355 nm excitation alongside the established UVC 266 nm source. The addition of 355 nm extends PARS contrast beyond 266 nm, enhancing stromal visualization (e.g., collagen, elastin) and capturing red blood cells, melanin, and other features through complementary radiative and non-radiative absorption. The 266 nm and 355 nm pulses interrogate the sample in an interlaced fashion, enabling concurrent acquisition without compromising imaging speed. Using the RegGAN image-translation framework, this work presents the first demonstration of PARS virtual staining across multiple specialized stains, including Masson's trichrome, periodic acid-Schiff (PAS), and Jones' silver, in addition to hematoxylin and eosin (H&E), across diverse human and murine tissues. A masked evaluation by expert pathologists showed that virtual stains achieved the same diagnostic quality as their chemical counterparts, and pathologists could not reliably distinguish real from virtual stains. By providing label-free multi-stain outputs from a single scan, dual-excitation PARS virtual staining could integrate into digital pathology workflows, expanding diagnostic utility. Real and virtual whole-slide image (WSI) pairs are publicly available at the BioImage Archive (https://doi.org/10.6019/S-BIAD2232).