Multimodal confocal mosaicing of basal cell carcinomas in Mohs surgical skin excisions

摘要

Mohs surgery is a procedure for microscopically excising basal cell carcinomas (BCCs) while preserving maximal surrounding normal skin. Each serial excision is guided by examination of the frozen histology of the previous excision. Because several (2-20) excisions must be made and frozen histology prepared for each excision. Mohs surgery is time-consuming (15-45 minutes per excision) and tedious. Real-time confocal reflectance mosaicing enables detection of BCCs directly in fresh excisions, following contrast-enhancement by acetowhitening. A confocal mosaic allows rapid observation of 15×15 mm~2 of tissue, which is equivalent to a low magnification, 2X view of the excision. Relatively large superficial nodular and micronodular BCCs are rapidly detectable in confocal reflectance mosaics, whereas detection of much smaller infiltrative and sclerosing BCCs is a challenge due to the lack of sufficient nuclear/dermis contrast in acetowhitened excisions. Multimodal contrast, combining reflectance with either fluorescence or autofluorescence may make it possible to detect infiltrative and sclerosing BCCs. A reflectance image shows both nuclei and the surrounding dermis, whereas an autofluorescence image (excitation at 488nm, detection 500-700nm) shows only the dermis. Thus, ability of a composite (i.e., reflectance-less-autofluorescence) image shows significantly darkened dermis, with stronger enhancement of nuclear/dermis contrast. Preliminary results illustrate that this may enable detection of infiltrative and sclerosing BCCs. The use of reflectance and autofluorescence parallels the use of two stains (hematoxylin and eosin) in histology, thus allowing a more complete optical detection method. Confocal microscopy of whole tissue samples shows promise to rapidly guide serial surgical excisions such as in Mohs surgery. Critical to success is the ability to preferentially contrast nuclei and tumors. Imaging modalities investigated toward this end included reflectance mode confocal microscopy (RCM) and fluorescence mode confocal microscopy (FCM). Exogenous fluorescent contrast agents included Acridine Orange and Eosin, which labeled the nucleus and cytoplasm respectively. Endogenous collagen autofluorescence highlighted the dermal component. Encorporating the fluorescence modality may help contrast tumors and nuclei amid bright dermal reflectance. Figure 1 shows a typical Basal cell carcinoma excision imaged with reflectance mode confocal microscopy and illustrates the need for additional contrast in the detection of small tumors.