Two photon microscopy and optical clearing for volumetric imaging of melanoma surgical margins

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Complete resection of melanoma is essential to patient survival, but tools for assessing the irregular borders of melanoma during surgery are rudimentary. Current surgical management emphasizes either multiple days of surgery with paraffin processing to evaluate margins or wide local excision that removes large margins of healthy tissue to minimize untreated residual disease. In either case, the need for large and disfiguring margins around melanoma stems from the diffuse and three-dimensional pattern of spread which is difficult to assess from two- dimensional histology. The goal of this R21 application is to develop a new approach to melanoma surgery based on comprehensive volumetric imaging of the tissue margin to determine the true extent of melanoma involvement. Using rapid molecular labeling, and two photon fluorescence microscopy combined with optical clearing (cTPFM), whole margins can be rendered clear and then imaged from the surface down to the level of tumor involvement, enabling complete assessment of tumor boundaries more rapidly and more accurately than current techniques such as rush paraffin sections. Following imaging, therapy can be directed to insufficient margins, potentially improving both tissue conservation and patient outcomes. This entirely new approach to melanoma therapy leverages previous experience in fluorescent labeling and proposes advances in rapid tissue clearing and new technology for high throughput imaging to clear and image specimens significantly faster than the current standard. The aims for this proposal are as follows: Aim 1, Task 1 will develop automated tissue clearing equipment that can reproducibly apply reagents in sequence to large numbers of specimens and conduct studies to establish non-interreference with histology. Task 2 will develop improved imaging equipment specifically for dermatologic surgery and clearing. Task 3 will develop and integrate fundamental advances in two photon fluorescence detection technology, enabling a 100-fold increase in imaging speed by scanning multiple depths in parallel. Aim 2, Task 1 will establish the sensitivity and specificity of TPFM by clearing and imaging historical melanoma specimens and then comparing histological evaluation of TPFM images to paraffin sections. The difference between conventional 2D and comprehensive 3D imaging will be evaluated in a blinded study. Task 2 will conduct exploratory intrasurgical imaging by recruiting patients being treated for melanoma and then clearing and imaging entire margins prior to histological processing, demonstrating that intrasurgical mapping of entire melanoma margins is feasible. Successful completion of this project would demonstrate a new approach to melanoma treatment that emphasizes tissue conservation, improves patient outcomes by detecting insufficient margins and reduces the need for complex surgical reconstruction.