In vivo reflectance confocal microscopy shows promise for the early detection of malignant melanoma (MM). Two hallmarks of MM have been identified: the presence of pagetoid melanocytes in the epidermis and the breakdown of the dermal papillae. For detection of MM, these
features must be identified qualitatively by the clinician and qualitatively through automated pattern recognition. A machine vision algorithm was developed for automated detection. The algorithm
detected pagetoid melanocytes and breakdown of the dermal/epidermal junction in a pre-selected set of five MMs and five benign nevi for correct diagnosis.
Mosaicing of confocal images enables observation of nuclear morphology in large areas of tissue. An application of interest is rapid detection of basal cell carcinomas (BCCs) in skin excisions during Mohs surgery. A mosaic is currently created in less than 9 min, whereas preparing frozen histology requires 20 to 45 min for an excision. In reflectance mosaics, using acetic acid as a contrast agent to brighten nuclei, large and densely nucleated BCC tumors were detectable in fields of view of 12×12 mm (which is equivalent to a 2×-magnified view as required by Mohs surgeons). However, small and sparsely nucleated tumors remained undetectable. Their diminutive size within the large field of view resulted in weak light backscatter and contrast relative to the bright surrounding normal dermis. In fluorescence, a nuclear-specific contrast agent may be used and light emission collected specifically from nuclei but almost none from the dermis. Acridine orange of concentration 1 mM stains nuclei in 20 s with high specificity and strongly enhances nuclear-to-dermis contrast of BCCs. Comparison of fluorescence mosaics to histology shows that both large and small tumors are detectable. The results demonstrate the feasibility of confocal mosaicing microscopy toward rapid surgical pathology to potentially expedite and guide surgery.
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
15x15 mm2 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.
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