There is a need to image excised tissues during tumor-resection procedures in order to identify residual tumors at the margins and to guide their complete removal. The imaging of dysregulated cell-surface receptors is a potential means of identifying the presence of diseases with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular-imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Here, we demonstrate that the topical application and quantification of a multiplexed cocktail of receptor-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) enables rapid quantitative molecular phenotyping (QMP) of the surface of freshly excised tissues to determine the presence of disease. In order to mitigate the ambiguity due to nonspecific sources of contrast such as off-target binding or uneven delivery, a ratiometric method is employed to quantify the specific vs. nonspecific binding of the multiplexed NPs. Validation experiments with human tumor cell lines, fresh human tumor xenografts in mice, and fresh human breast specimens demonstrate that QMP imaging of excised tissues agrees with flow cytometry and immunohistochemistry, and that this technique may be achieved in less than 15 minutes for potential intraoperative use in guiding breast-conserving surgeries.

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.

Line-scanning, with pupil engineering and the use of linear array detectors, may enable simple, small and low-cost confocal microscopes for clinical imaging of human epithelial tissues. However, a fundamental understanding of line-scanning performance within the highly scattering and aberrating conditions of human tissue is necessary, to translate from benchtop instrumentation to clinical implementation. The results of a preliminary investigation for reflectance imaging in skin are reported.Point-scanning confocal microscopes are well-proven for optical sectioning and imaging of nuclear and cellular detail in human skin [1]. However, line-scanning is fundamentally simpler, and the recent advent of high-quality linear array detectors may enable smaller and lower-cost confocal microscopy [2][3][4]. Scanning only once, directly behind the pupil of the objective lens, and de-scanning once, directly onto a linear array detector, allows considerable simplification of the original confocal line-scanning designs.Of particular clinical importance is imaging performance with reflectance as an endogenous source of contrast, in highly scattering and aberrating human tissues. A fundamental understanding of confocal line-scanning performance in human tissue is therefore a necessary translational bridge from benchtop instrumentation to clinical implementation. For such investigations, the epidermis in human skin is easily accessible and available, and is an excellent model for the scattering and aberrating properties of epithelial tissue. In this Letter, we present a full-pupil line-scanning confocal microscope, and comparison of its performance to a divided-pupil configuration, for reflectance imaging of nuclear and cellular morphology in human epidermis, both in vivo and ex vivo.As in the divided-pupil design that we reported earlier [5,6], the full-pupil line-scanning microscope, too, consists of 8-10 optical components in a simple configuration, with total hardware costs of $15,000. The electronics is based on field programmable gate array (FPGA) logic. The development of FPGA-based electronics offers the advantages of small footprint, rapid reconfigurability and complete integration toward a stand-alone system. In the long-term, line-scanning with the use of FPGA-based electronics may enable simple, small, low-cost stand-alone confocal microscopes for use at-the-bedside in diverse healthcare settings worldwide. Figure 1 shows the full-pupil line-scanning confocal microscope. A collimated beam is incident on a cylindrical lens (L cyl ), which is placed so as to produce a focused line in the back focal plane (BFP) of an objective lens (Obj). The focused line in the BFP is Fourier-transformed or re-focused to a line that is oriented perpendicular to the axis of the cylindrical lens. This produces a focused line in the object plane (within tissue) that is oriented perpendicular to the plane of this page. The line is scanned by an oscillating galvanometrically-driven scan mirror (G). The scan mirror is placed as close ...