Feasibility of digitally stained multimodal confocal mosaics to simulate histopathology

Abstract

Fluorescence confocal mosaicing microscopy of tissue biopsies stained with acridine orange has been shown to accurately identify tumors and with an overall sensitivity of 96.6% and specificity of 89.2%. However, fluorescence shows only nuclear detail similar to hematoxylin in histopathology and does not show collagen or cytoplasm, which may provide necessary negative contrast information similar to eosin used in histopathology. Reflectance mode contrast is sensitive to collagen and cytoplasm without staining. To further improve sensitivity and specificity, digitally stained confocal mosaics combine confocal fluorescence and reflectance images in a multimodal pseudo-color image to mimic the appearance of histopathology with hematoxylin and eosin and facilitate the introduction of confocal microscopy into the clinical realm.

Figure 1

(A–D) 2X magnification. The reflectance mosaic (a) is colored pink and the fluorescence mosaic (b) is colored purple in the DSCP image (c). The correlating H&E histopathology (d) is also shown at 2X magnification. The arrows (c) indicate a micronodular focus in the deeper dermis and the superficial epidermis. (e–j) 30X magnification. A micronodular tumor focus in the dermis (e) and normal superficial epidermis (f) are magnified from figure 1c. In a separate sample containing infiltrative collagen is bright in the deeper dermis in reflectance mode (g). The correlating fluorescence image (h) shows bright nuclei. The DSCP image (i) is shown with the correlating histopathology (j).