Label-free whole slide virtual multi-staining using dual-excitation photon absorption remote sensing microscopy

Histochemical staining is essential for visualizing tissue architecture but is destructive and limited by tissue availability. Virtual staining with label-free microscopy offers a non-destructive alternative, enabling multiple stains from the same section. We present a dual-excitation Photon Absorption Remote Sensing (PARS) microscopy system, the first application of 355 nm UVA alongside 266 nm UVC excitation. The 355 nm source extends PARS contrast to include red blood cells, melanin, and enhanced stromal architecture through complementary radiative and non-radiative absorption. Using the RegGAN framework, we demonstrate the first PARS virtual staining across routine (H&E) and specialized (Masson’s trichrome, PAS, Jones methenamine silver) stains in human and murine tissues. Quantitative metrics show dual-excitation improves virtual stain similarity over single-wavelength inputs. A masked evaluation by expert pathologists provides an initial, coarse assessment indicating that virtual stains achieve ratings on a visual diagnostic quality scale comparable to chemical counterparts under limited evaluation conditions. These results support dual-excitation PARS as a promising non-destructive approach for multi-stain virtual histology. Corresponding whole-slide image pairs are available at the BioImage Archive (10.6019/S-BIAD2232).