Precise removal of basal cell carcinomas (BCCs) with minimal damage to the surrounding normal skin is guided by the examination of frozen histology of each excision during Mohs surgery. The preparation of frozen histology is slow, requiring 20 to 45 min per excision. Confocal reflectance mosaicing may enable rapid detection of BCCs directly in surgical excisions, with minimal need for frozen histology. Soaking the excisions in acetic acid rapidly brightens nuclei and enhances BCC-to-dermis contrast. Clinically useful concentrations of acetic acid from 10 to 1% require 30 s to 5 min, respectively. A tissue fixture precisely controls the stability, flatness, tilt, and sag of the excisions, which enables mosaicing of 36x36 images to create a field of view of 12x12 mm. This simulates a 2x magnification view in light microscopes, which is routinely used by Mohs surgeons to examine frozen histology. Compared to brightfield, cross-polarization enhances contrast and detectability of BCCs in the papillary dermis but not in the reticular dermis. Comparison of mosaics to histology shows that nodular, micronodular, and superficial BCCs are easily detected. However, infiltrative and sclerosing BCCs tend to be obscured within the surrounding bright dermis. The mosaicing method currently requires 9 min, and thus may expedite Mohs surgery.
SummaryPrecise micro-surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour-intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10-1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5-5 min and enhances nuclear-to-dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 × 36 images to create a field of view of 12 × 12 mm. This large field of view displays the excision at 2× magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20-45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology-at-the-bedside to expedite and guide surgery.
A system was designed and developed for simultaneous fluorescence and reflectance contrast in vivo confocal imaging of murine skin using 488 nm (fluorescence mode) and 830 nm (reflectance mode) laser light sources. B16 melanoma cells and B16-enhanced green fluorescent protein (EGFP) cells were inoculated intradermally into transgenic C57BL/6-TgN (ACTbEGFP) 10sb and non-transgenic C57BL/6 mice, respectively. The inoculation sites were imaged sequentially over a 20 d period. The in vivo confocal images were correlated with ex vivo conventional microscopy. The combined modality system provided single-cell resolution and adequate image registration. In fluorescence mode, B16 melanoma cells appeared as dark objects in the bright background of the GFP expressing murine cells of the C57BL/6 transgenic mouse, and the B16-EGFP melanoma cells had a bright signal within a dark background in C57BL/6 mice. In the C57BL/6 transgenic mouse, a population of fluorescent dendritic cells was observed in the vicinity of the tumor cells. The reflectance images provide a useful reference for those areas in the dermal tissues lacking a fluorescent signal. Combined reflectance/fluorescence in vivo confocal laser scanning microscopy holds significant promise for studies of tumor progression in murine skin.
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