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

黑色素瘤的完全切除对患者的生存至关重要，但评估黑色素瘤不规则边界的工具 手术中的黑色素瘤是初步的。目前的手术治疗强调的是多天的 石蜡处理以评估边缘的手术或广泛的局部切除， 组织，以尽量减少未经治疗的残留疾病。在这两种情况下，需要大量和毁容的利润率左右， 黑色素瘤源于扩散和三维模式，其难以从二维评估， 三维组织学这项R21应用的目标是开发一种新的黑色素瘤手术方法 基于组织边缘的全面体积成像来确定 黑色素瘤参与。使用快速分子标记和双光子荧光显微镜 结合光学清除（cTPFM），整个边缘可以清晰呈现，然后从 表面下至肿瘤累及水平，能够更快地对肿瘤边界进行全面评估 并且比目前的技术如灯心草石蜡切片更精确。成像后，治疗可以是 针对不足的边缘，潜在地改善组织保护和患者结果。这 黑色素瘤治疗的全新方法利用了荧光标记的先前经验， 提出了快速组织清除和高通量成像新技术的进展，以清除和成像 样品的速度明显快于目前的标准。本提案的目标如下：目标1，任务 1将开发自动化组织清除设备，该设备可以重复地将试剂依次应用于大的 样本数量，并进行研究，以建立与组织学的非相互参照。任务2将开发 专门用于皮肤外科手术和清理的改进的成像设备。任务3将开发和整合 双光子荧光检测技术的根本性进步，使成像能力提高100倍 通过并行扫描多个深度来提高速度。目标2，任务1将建立TPFM的敏感性和特异性 通过清除和成像历史黑色素瘤标本，然后比较TPFM的组织学评价， 图像到石蜡切片。传统的2D和全面的3D成像之间的区别将是 在盲法研究中进行评估。任务2将通过招募患者进行探索性术中成像 进行黑色素瘤治疗，然后在组织学处理之前对整个边缘进行清除和成像， 这表明整个黑素瘤边缘的手术内定位是可行的。成功完成本 该项目将展示一种新的黑色素瘤治疗方法，强调组织保护， 通过检测边缘不足改善患者结局，并减少复杂手术的需要 重建

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.