Abstract
Conventional histopathological techniques, such as hematoxylin and eosin staining, are limited to 4-5 μm-thick tissue sections, restricting visualization to two-dimensional planes. Moreover, acquisition of three-dimensional horizontal images from the skin surface remains challenging, hindering precise assessment of tumor margins in skin lesions. This challenge is particularly pronounced in extramammary Paget's disease (EMPD), in which diffuse epidermal tumor cell spread complicates accurate evaluation of lesion extent. We hypothesized that combining horizontal sectioning with identification of individual tumor cells would enhance the determination of surgical margins. In this study, we developed a deep-imaging technique utilizing fluorescent solvatochromic dyes (LipiORDER(®) and HistoBright(®)) and two-photon microscopy to achieve high-resolution tumor margin visualization in EMPD. This technique enables identification of tumor cells in frozen and paraffin-embedded tissue blocks, as well as in live skin tissue under physiological conditions. Our novel approach holds substantial promise for improving the precision of surgical-margin assessment in EMPD and other cutaneous malignancies.