Histopathology for Mohs micrographic surgery with photoacoustic remote sensing microscopy

Abstract

Mohs micrographic surgery (MMS) is a precise technique where layers of tissue are resected and examined with intraoperative histopathology to minimize the removal of normal tissue while completely excising the cancer. To achieve intraoperative pathology, the tissue is frozen, sectioned and stained over a 20-to 60-minute period, then analyzed by the MMS surgeon. Surgery is continued one layer at a time until no cancerous cells remain, meaning MMS can take several hours to complete. Ideally, it would be desirable to circumvent or augment frozen sectioning methods and directly visualize subcellular morphology on the unprocessed excised tissues. Employing photoacoustic remote sensing (PARS™) microscopy, we present a non-contact label-free reflection-mode method of performing such visualizations in frozen sections of human skin. PARS leverages endogenous optical absorption contrast within cell nuclei to provide visualizations reminiscent of histochemical staining techniques. Here, we demonstrate the ability of PARS microscopy to provide large grossing scans (>1 cm 2 , sufficient to visualize entire MMS sections) and regional scans with subcellular lateral resolution (400 ± 150 nm).

Single acquisition label-free histology-like imaging with dual-contrast photoacoustic remote sensing microscopy

“… 15 – 21 These techniques have subsequently provided hematoxylin-like visualizations of nuclear structures in tissue blocks, slides, and fresh tissues. 15 – 21 However, these techniques do not provide full H&E emulation, as they lack the extranuclear tissue structures captured by eosin staining. Here, we propose approaching this problem by leveraging, for the first time, PARS unique dual scattering and absorption contrast.…”

“…Therefore, this system provides H&E emulation with imaging rates comparable to previously reported UV excitation PARS systems. 21 The current implementation of the system is capable of collecting high-resolution images with 250-nm spatial sampling at a rate of ( for an area equivalent to that shown in Fig. 3 ).…”

“…These samples were previously used to provide the first direct comparison between PARS and H&E imaging. 21 As outlined in the corresponding publication, unstained frozen skin tissue sections were collected and scanned with the PARS microscope. 21 Following PARS imaging, the unstained sections were stained and imaged with a standard light microscope.…”

“… 21 As outlined in the corresponding publication, unstained frozen skin tissue sections were collected and scanned with the PARS microscope. 21 Following PARS imaging, the unstained sections were stained and imaged with a standard light microscope. 21 The corresponding H&E and PARS visualizations of a small region of this tissue are shown in Figs.…”

“… 15 – 18 Applied to histological imaging, PARS has successfully used UV excitation to capture nuclear structures. 15 – 21 This technique is efficacious in providing hematoxylin-like contrast in frozen sections and paraffin-embedded blocks and sections. 15 – 21 However, these UV-PARS visualizations lack some essential diagnostic details.…”