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

完全切除黑色素瘤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基本的。目前的外科治疗强调要么是多天的 用石蜡处理的手术评估切缘或广泛的局部切除切除健康的大切缘 以最大限度地减少未治疗的残留病。在任何一种情况下，都需要巨大且令人难堪的利润率 黑色素瘤起源于弥漫的和三维的扩散模式，这种模式很难从两个方面进行评估- 空间组织学。此R21应用程序的目标是开发一种治疗黑色素瘤的新方法 根据组织边缘的综合体积成像来确定真实范围 黑色素瘤牵连。使用快速分子标记和双光子荧光显微镜 与光学清理(CTPFM)相结合，可以使整个页边距变得清晰，然后从 从表面到肿瘤受累程度，能够更快地完成对肿瘤边界的评估 而且比目前的技术，如冲刷石蜡切片更准确。在成像之后，治疗可以 针对不足的利润率，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以前在荧光标记和 提出了快速组织清除的进展和高通量成像的新技术 样品的速度明显快于现行标准。这项提议的目标如下：目标1，任务 1将开发自动组织清理设备，可以重复地按顺序将试剂应用到大型 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将制定 改进了专门用于皮肤科手术和清理的成像设备。任务3将开发和集成 双光子荧光探测技术的根本性进展，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将确定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的组织学评估 图像到石蜡切片。常规2D成像和全面3D成像之间的区别将是 在一项盲目研究中进行评估。任务2将通过招募患者进行探索性手术内成像 治疗黑色素瘤，然后在组织学处理之前清除和成像整个边缘， 证明了整个黑色素瘤边缘的手术内标测是可行的。成功完成这项工作 该项目将展示一种强调组织保护的黑色素瘤治疗新方法， 通过检测边缘不足来改善患者预后，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.