Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12  mm² of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10  mm² of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10  mm² of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5  cm² piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

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Background Fluorescence confocal mosaicing microscopy is an emerging technology for rapid imaging of nuclear and morphologic detail directly in excised tissue, without the need for frozen or fixed section processing. Basal cell carcinomas (BCCs) can be detected with high sensitivity and specificity in Mohs excisions with this approach. For translation to clinical trials and toward potentially routine implementation, a new and faster approach called strip mosaicing confocal microscopy was recently developed. Objectives To perform a preliminary assessment of fluorescence strip mosaicing confocal microscopy for detecting skin cancer margins in Mohs excisions. Methods Tissue from 17 Mohs cases was imaged in the form of strip mosaics. Each mosaic was divided into two halves (submosaics) and graded by a Mohs surgeon, who was blinded to the pathology. The 34 submosaics were compared to the corresponding Mohs pathology. Results The overall image quality was excellent for resolution, contrast and stitching in the 34 submosaics. Components of normal skin including the epidermis, dermis, dermal appendages and subcutaneous tissue were easily visualized. Preliminary measure of sensitivity and specificity was 94% for detecting skin cancer margins. Conclusions The new strip mosaicing approach represents another advance in confocal microscopy for imaging of large areas of excised tissue. Strip mosaicing may enable rapid assessment of BCC margins in fresh excisions during Mohs surgery and may serve as an adjunct for frozen pathology.

Abstract. Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen-or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750 × 750 μm 2 . We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2 × 2 cm 2 of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant.

Point-scanning reflectance confocal microscopes continue to be successfully translated for detection of skin cancer. Line-scanning, with the use of a single scanner and a linear-array detector, offers a potentially smaller, simpler and lower cost alternative approach, to accelerate widespread dissemination into the clinic. However, translation will require an understanding of imaging performance deep within scattering and aberrating human tissues. We report the results of an investigation of the performance of a full-pupil line-scanning reflectance confocal microscope in human skin and oral mucosa, in terms of resolution, optical sectioning, contrast, signal-to-noise ratio, imaging and the effect of speckle noise.

Abstract. For the removal of superficial and nodular basal cell carcinomas (BCCs), laser ablation provides certain advantages relative to other treatment modalities. However, efficacy and reliability tend to be variable because tissue is vaporized such that none is available for subsequent histopathological examination for residual BCC (and to confirm complete removal of tumor). Intra-operative reflectance confocal microscopy (RCM) may provide a means to detect residual tumor directly on the patient and guide ablation. However, optimization of ablation parameters will be necessary to control collateral thermal damage and preserve sufficient viability in the underlying layer of tissue, so as to subsequently allow labeling of nuclear morphology with a contrast agent and imaging of residual BCC. We report the results of a preliminary study of two key parameters (fluence, number of passes) vis-à-vis the feasibility of labeling and RCM imaging in human skin ex vivo, following ablation with an erbium:yttrium aluminum garnet laser. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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