Comparison of nonlinear microscopy and frozen section histology for imaging of Mohs surgical margins

Abstract

Mohs surgery uses en face frozen section analysis (FSA) with complete margin examination for the excision of select basal cell carcinomas (BCC), obtaining excellent cosmetic outcomes and extremely low recurrence rates. However, Mohs with FSA is time-consuming because of the need to iteratively perform cryosectioning on sequential excisions. Fluorescent microscopies can image tissue specimens without requiring physical sectioning, potentially reducing the time to perform Mohs surgery. We demonstrate a protocol for nonlinear microscopy (NLM) imaging of surgical specimens that combines dual agent staining, virtual H&E rendering, and video rate imaging. We also introduce a novel protocol that enables micron-level co-registration of NLM images with FSA histology, and demonstrate that NLM can reproduce similar features similar to FSA in BCC specimens with both negative and positive surgical margins. We show that the fluorescent labels can be extracted with conventional vacuum infiltration processing, enabling subsequent immunohistochemistry on fluorescently labeled tissue. This protocol can also be applied to evaluate the performance of NLM compared with FSA in a wide range of pathologies for intraoperative consultation.

Fig. 1.

Method used to NLM image Mohs surgical specimens (red) with precise registration to FSA histology. A) Saucer-shaped tissue specimen is removed during surgery. B) Relaxing incisions are made to partially flatten the tissue. C) The tissue is fully flattened against a heat extractor and frozen in optimal cutting temperature (OCT) media. D) The tissue is cryosectioned to produce an FSA slide for margin evaluation. E) At this stage the block is no longer required for diagnosis and is discarded. An additional FSA section and a thicker volume of frozen tissue are sectioned for study immediately adjacent to the final FSA plane used during margin evaluation. F) Thawing the frozen block distorts the bulk tissue, but there is minimal distortion of the section used for NLM imaging.

Fig. 2.

Normal skin removed during Mohs surgery. High magnification of adipose tissue (green inset), shows adipocytes with red surgical marking ink. Fat is difficult to freeze and appears distorted in FSA histology, while NLM better represents the original tissue appearance. Higher magnification views of the epidermis (blue) show normal structure with sebaceous glands, hair follicles and a hidrocystoma. Full resolution: http://imstore.mit.edu/mohs-boe/Fig2/Fig2.html.

Fig. 3.

Mohs margin with mixed nodular and infiltrative pattern BCC. Red surgical marking ink is visible along the top of the figure (red arrows) in both the NLM and FSA images, while green surgical ink (green arrow), which lacks fluorescence, is visible only in the FSA image. Full resolution: http://imstore.mit.edu/mohs-boe/Fig3/Fig3.html.

Fig. 4.

Excision with large nodular BCC. The FSA section intercepts the edge of the tumor, while the NLM image is from a section tens of microns further into the tumor, showing a larger area of nodular BCC. At low magnification, multiple nodules of basaloid tumor with peripheral palisading form a large deep dermal tumor mass. At higher magnification, palisading of tumor cells at the periphery of the nodule and peritumoral stromal mucin, both characteristics of basal cell carcinomas, are seen equally well on NLM and FSA images. Full resolution: http://imstore.mit.edu/mohs-boe/Fig4/Fig4.html.

Fig. 5.

Mohs excision with a large mass of infiltrative CC. The central portion of each half of the excision contains nests and cords of tumor cells that irregularly infiltrate into the deep fatty subcutaneous tissue. The overlying epidermis is histologically normal. Full resolution: http://imstore.mit.edu/mohs-boe/Fig5/Fig5.html.

Fig. 6.

Mohs excision with well differentiated squamous cell carcinoma (SCC). The high magnification view (bottom) shows a region of invasion. http://imstore.mit.edu/mohs-boe/Fig6/Fig6.html.

Fig. 7.

SHG (left column) and AO/SR101 fluorescence NLM imaging (center column) image of fresh human skin (not frozen) both before (top row) and after (bottom) fixation and processing on a vacuum infiltration processor. SHG is almost completely unaffected by vacuum infiltration processing because it is an intrinsic signal produced by collagen, which is not soluble in either ethanol or xylene. In contrast, the highly soluble AO and SR101 labels are reduced to undetectable levels after processing. In the right column, a magnified view of a sebaceous gland with SHG (blue), AO (red) and SR101 (green) shows the collagen is unchanged after vacuum processing, but the fluorescent signal from cell nuclei is reduced to undetectable levels.

Fig. 8.

Demonstration of IHC on tissue after AO/SR101 labelling and washout. Melan-A (left panel) highlights the cytoplasm of an isolated melanocyte within the sebaceous gland, with a high signal and without non-specific labeling of other cell types. Similarly, we observed high signal and specific cytokeratin labeling of the basal layer is a sebaceous lobule (right panel). These results are consistent with the finding that AO/SR101 are extracted from tissue.