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

项目摘要 多光子显微镜（MPM）可以提供亚微米分辨率的活体组织在其自然状态下的图像， 环境与无标记的分子对比从多种形式，包括二次谐波产生 (SHG)和双光子激发荧光（TPEF）。几种内源性组织成分可以是 可视化，包括胶原蛋白（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）。我们有先进的无标签 皮肤临床/转化研究中的MPM技术，用于表征角质细胞代谢，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， 并评估皮肤激光治疗的效果。其中许多研究都是使用 用于临床皮肤成像的商用多光子显微镜 (FOV)速度、占地面积和成本。为了解决这些临床采用的障碍，我们建议建立 “下一代”临床多光子显微镜，将先进的台式技术集成到 紧凑、实用、经济实惠的床边设备。这种新仪器将具有可比的FOV， 分辨率和扫描功能的标准护理反射共焦显微镜（RCM），但提供 多种模式（TPEF和SHG）的独特结构和代谢对比， 关于MPM我们将在一项轻剂量递增研究中确定该器械的临床安全性， DNA和细胞损伤，并在一项12例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，我们将进行伤口上皮再生的试点研究 以及白癜风微移植背景下的黑素细胞迁移， 移植到白癜风患者的皮肤中，这种皮肤缺乏黑色素细胞。黑素细胞迁移出 移植的皮肤和移植皮肤内翻转的角质形成细胞可以通过测量TPEF而可视化 细胞黑色素和辅助因子（NADH，FAD+）在移植物中和周围，有效地识别不同的细胞 参与伤口愈合的人群。我们广泛的，长期的目标是发展ev-MPM作为一个实用的 一种快速体内表征细胞形态和代谢成像终点的方法， 患者这些可以用来了解和优化伤口愈合，并提供一个实用的旁边的平台 用于检测、诊断和优化皮肤疾病的治疗反应。

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.