Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue.

The accurate and complete removal of tumor, with minimal removal of healthy surrounding tissue, is guided by the examination of pathology. The preparation of pathology, either during surgery or after, is time-consuming and labor-intensive. In the setting of nonmelanoma skin cancers, frozen pathology that is prepared during Mohs surgery requires 20 to 45 min per excision, and two or more excisions are performed, such that the total preparation time lasts from two to several hours.1 In other settings such as with head-and-neck and breast cancer, fixed pathology is prepared after surgery. Preparation of fixed sections requires one to two days. Since it takes such a long time to obtain pathology results, the patient is sent home immediately after surgery. If the pathology shows positive tumor margins, the patient must undergo additional surgery (resection), and/or radiotherapy or chemotherapy. The incomplete removal of tumor and positive margins are reported to occur in 20 to 70% of patients, depending on the surgical setting.2,3 Although the rates of resections can be reduced by aggressive removal of tissue from a wide margin around the tumor, it can affect the functionality of the organ or produce unacceptable aesthetical results for the patient. Therefore the objective is to minimize the removal of healthy tissue by reducing the margins and still perform complete removal of the tumor. To address this problem, high-resolution optical imaging methods capable of displaying nuclear and cellular morphology, offer the possibility for rapid detection of tumors in large areas of freshly excised or biopsied tissue. Currently, confocal microscopy, full-field optical coherence tomography, multispectral macro-imaging, multiphoton microscopy, fluorescence lifetime imaging and other approaches are being developed for this purpose.4–16,17 We have been developing confocal mosaicing microscopy as a technology platform for imaging tumor margins in fresh tissue excisions from surgery2,3,13–15 to provide high-resolution images of large areas of tissue within minutes. Previously, we collected square-shaped images with aspect ratios of ∼1∶1, and stitched them together with custom software into a mosaic to display a large field of view. Mosaicing of 36-×-36 images to display 12-×-12  mm2 of excised tissue from Mohs surgery was demonstrated in 9 min.13–15 In a blinded examination of 45 fluorescence mosaics by two Mohs surgeons, basal cell carcinoma margins were detected with an overall sensitivity of 96.6% and a specificity of 89.2%.18,19 The results of this preclinical study demonstrated the promise of confocal mosaicing microscopy. Although 9 min is certainly faster than the hours or days required for preparing conventional (frozen or fixed) pathology, routine implementation in surgical settings will require shorter processing times. The unequivocal feedback from surgeons was that this technology was unlikely to be adopted unless the speed was improved. The clear expectation was that to reach practical and routine use in any operating room setting, mosaicing must meet the surgeons’ need to examine tumor margins in large areas (∼cm2) within a few minutes. To address this need we designed a faster approach called “strip mosaicing.” The initial instrumentation work for strip mosaicing was reported last year.20 Strip mosaicing is performed with a combination of optical and mechanical scanning. The tissue is mechanically translated across a linearly scanned focused laser beam. This approach is faster because the speed of mosaicing strips is primarily governed by the fast line acquisition rate in the confocal microscope20 and does not use the single frame acquisition method previously reported.14,18 In this paper, we report further advances and enhancement of the electronics and mechanics, the development of custom software, and the overall integration of hardware and software into an approach that demonstrates imaging of 1-×-1  cm2 of human skin tissue with 1-μm lateral resolution in 90 s and 5.5-×-3.5  cm2 of breast tissue with 0.8-μm lateral resolution in 13 min. The enhancements include a device to mount and flatten fresh tissue from surgery, improvements in the tissue translation stage for speed, accuracy and precision, synchronization of the optical and mechanical scanning to optimize alignment among and registration of strips, and stitching of image strips in parallel with acquisition using custom software.