Development of a fast scanning, extended field-of-view multiphoton microscope for clinical skin imaging

项目总结 多光子显微镜(MPM)可以提供活组织的亚微米分辨率图像 具有来自多种模式的无标记分子对比的环境，包括二次谐波产生 和双光子激发荧光(TPEF)。几种内源性组织成分可以是 可视化，包括胶原(来自SHG)和还原型烟酰胺腺嘌呤二核苷酸(NADH)，黄素 腺苷二核苷酸(FAD)、角蛋白、黑色素和弹性蛋白纤维(来自TPEF)。我们有先进的免标签 MPM技术在皮肤临床/转化学研究中用于角质形成细胞的代谢、诊断 黑色素瘤，了解黑素细胞生物学，检测基底细胞癌，量化皮肤色素沉着， 以及评估皮肤激光治疗的效果。这些研究中的许多都是使用 用于临床皮肤成像的商用多光子显微镜在视野方面有局限性 (FOV)、速度、占用空间和成本。为了解决临床采用的这些障碍，我们建议建立 将先进的台式技术集成到一个 紧凑、实用、经济实惠的床边设备。这种新的仪器将有类似的视场， 分辨率和扫描功能的标准反射共焦显微镜(RCM)，但提供 多种模式(TPEF和SHG)的独特结构和代谢对比只能实现 使用MPM。我们将在一项小剂量升级研究中确定该设备的临床安全性，该研究评估 DNA和细胞损伤，并在12名健康患者的临床研究中建立关键性能基准 不同皮肤类型的志愿者。此外，我们还将进行伤口再上皮化的初步研究。 以及白癜风微型移植物中的黑素细胞迁移，这是一种临床程序，其中有色皮肤是 移植到没有黑素细胞的白癜风患者皮肤上。黑素细胞迁移出 移植皮肤和角质形成细胞在移植皮肤内的翻转可以通过测量TPEF来可视化。 移植物内和周围的细胞黑色素和辅助因子(NADH、FAD)的变化，有效地识别不同的细胞 参与伤口愈合的人群。我们广泛的、长期的目标是将EV-MPM作为一种实用的 快速、活体表征细胞形态和代谢成像终点的方法 病人。这些都可以用来了解和优化伤口愈合，并提供一个实用的旁边平台 用于检测、诊断和优化皮肤病的治疗反应。

PROJECT SUMMARY Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native environment with label-free molecular contrast from multiple modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). Several endogenous tissue components can be visualized, including collagen (from SHG) and reduced nicotinamide adenine dinucleotide (NADH), flavin adenosine dinucleotide (FAD), keratin, melanin and elastin fibers (from TPEF). We have advanced label-free MPM technologies in skin clinical/translational studies for characterizing keratinocyte metabolism, diagnosing melanoma, understanding melanocyte biology, detecting basal cell carcinoma, quantifying skin pigmentation, and assessing the effects of cutaneous laser therapy. Many of these studies have been completed using a commercial multi-photon microscope for clinical skin imaging that has limitations in terms of field-of-view (FOV), speed, footprint, and cost. In order to address these barriers to clinical adoption, we propose to build a “next-generation” clinical multiphoton microscope that integrates advanced benchtop technologies into a compact, practical, and cost-effective bedside device. This new instrument will have comparable FOV, resolution, and scanning features to standard-of-care reflectance confocal microscopes (RCM), yet provide unique structural and metabolic contrast from multiple modalities (TPEF and SHG) that can only be achieved with MPM. We will establish the clinical safety of this device in a light dose escalation study that assesses DNA and cellular damage, and establish key performance benchmarks in a 12-patient clinical study of healthy volunteers across a range of skin types. In addition, we will conduct pilot studies of wound re-epithelialization and melanocyte migration in the context of vitiligo micro-grafts, a clinical procedure where pigmented skin is transplanted into skin affected by vitiligo, which is devoid of melanocytes. Melanocytes migrating out of engrafted skin and keratinocytes turning over within engrafted skin can be visualized by measuring the TPEF of cellular melanin and co-factors (NADH, FAD+) in and around the grafts, effectively identifying different cell populations involved in wound healing. Our broad, long term goal is to develop ev-MPM as a practical approach for rapid, in vivo characterization of cellular morphologic and metabolic imaging endpoints in patients. These can be used to understand and optimize wound healing and provide a practical beside platform for detecting, diagnosing, and optimizing therapeutic response in skin diseases.