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

双光子显微镜和光学透明技术用于黑色素瘤手术切缘的体积成像

• 批准号: 10553713
• 负责人: Michael Gene Giacomelli
• 金额: $ 14.86万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553713
• 关键词: 3-Dimensional; Acceleration; Area; Assessment tool; Blinded; Clinical; Collection; Complex; Computer software; Consent; Custom; Day Surgery; Dermatologic; Detection; Diagnostic; Diagnostic Neoplasm Staging; Diffuse; Diffusion; Distant Metastasis; Equipment; Evaluation; Excision; Fluorescence; Fluorescence Microscopy; Frozen Sections; Future; Goals; Guidelines; Head; Histologic; Histology; Histopathology; Hour; Human; Image; Image-Guided Surgery; Infiltration; Intervention Studies; Investigational Therapies; Label; Malignant Neoplasms; Maps; Medical center; Methods; Microscope; Minority; Mohs Surgery; Molecular; Operative Surgical Procedures; Optics; Paraffin; Pathology; Patient Recruitments; Patient-Focused Outcomes; Patients; Pattern; Postoperative Period; Precancerous melanosis; Procedures; Radial; Reagent; Reconstructive Surgical Procedures; Reproducibility; Residual Cancers; Residual Neoplasm; Resolution; Resources; Risk; Risk Reduction; Scanning; Sensitivity and Specificity; Silicon; Skin; Skin Cancer; Specimen; Speed; Staging; Stains; Sun Exposure; Surface; Surgeon; Surgical Management; Surgical margins; Survival Rate; Techniques; Technology; Temperature; Testing; Thick; Three-Dimensional Imaging; Time; Tissue imaging; Tissues; Universities; Visualization; Work; cancer imaging; cancer surgery; clinical practice; cost; digital pathology; experience; experimental study; first-in-human; follow-up; high risk; high throughput technology; histological image; improved; melanoma; microscopic imaging; millimeter; new technology; novel strategies; photomultiplier; preservation; pressure; stem; tumor; two photon microscopy; two-dimensional; two-photon

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.

黑色素瘤的完全切除对于患者的生存至关重要，但评估黑色素瘤不规则边界的工具 手术期间的黑色素瘤是初级的。目前的手术治疗强调多天 使用石蜡处理来评估边缘的手术或广泛的局部切除术，去除健康的大边缘 组织以尽量减少未经治疗的残留疾病。无论哪种情况，都需要在周围留出大的、损毁外观的边距。 黑色素瘤源于弥漫性和三维扩散模式，很难从二维角度进行评估 三维组织学。 R21 应用的目标是开发一种新的黑色素瘤手术方法 基于组织边缘的综合体积成像来确定真实的范围 黑色素瘤参与。使用快速分子标记和双光子荧光显微镜 与光学清除 (cTPFM) 相结合，可以使整个边缘变得清晰，然后从 表面深入到肿瘤受累的水平，从而能够更快地完整评估肿瘤边界 并且比目前的技术（例如灯芯石蜡切片）更准确。影像学检查后，可以进行治疗 针对边缘不足的问题，有可能改善组织保存和患者预后。这 黑色素瘤治疗的全新方法利用了以前的荧光标记经验和 提出了快速组织透明化和高通量成像新技术的进步，以实现清晰和成像 样本的采集速度明显快于现行标准。该提案的目标如下：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按顺序应用于大型组织 数量的标本并进行研究以确定与组织学的不相关性。任务2将展开 改进了专门用于皮肤手术和清理的成像设备。任务3将开发和整合 双光子荧光检测技术取得根本性进步，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。传统2D和全面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 接受黑色素瘤治疗，然后在组织学处理之前对整个边缘进行清理和成像， 证明对整个黑色素瘤边缘进行术中测绘是可行的。顺利完成本次 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的治疗效果，并减少复杂手术的需要 重建。