Super resolution label-free imaging in highly scattering bone

项目摘要 间充质干细胞（也称为间充质基质细胞或MSC）具有很大的前景， 由于其旁分泌、多潜能和免疫抑制作用，可用于治疗骨相关疾病。一 主要目的是利用MSC的成骨分化作为代谢性骨的潜在治疗方法 疾病（如骨质疏松症、骨生成障碍或磷酸酶过少症）或局部骨缺损。 然而，MSC分化控制途径的知识以及如何在分化过程中利用这些途径， 缺乏生物处理，并且阻碍了更广泛的临床预防1 - 3。可惜现在的 用于表征MSC效力的临床和工业现有技术是简单地测量一些表面 使用流式细胞术检测细胞标志物，并研究细胞在体外分化成靶组织（例如， 骨/软骨）。这些测定具有定量挑战性，执行缓慢，并且与免疫原性的直接相关性低。 体内功能4.我们的提案旨在利用新型成像技术的发展来识别 对移植干细胞分化和健康骨组织具有关键意义的体内过程 生产，并将询问和利用膜脂途径，以维持和提高 MSC的分化潜能。 我们计划的研究的动机是越来越多的赞赏如何脂膜加工 调节在骨形成中具有中心调节重要性的细胞特性， 在直径约500纳米的多泡体中产生并分泌矿化基质囊泡， 使骨胶原中羟基磷灰石矿物晶体的沉积和生长成核5，6.知之甚少 驱动这些囊泡的形成和分泌的过程，这是一个至关重要的途径， 成熟软骨和骨骼的形成。这个形成过程是具有挑战性的标签，但与高 折射率变化，它是理想的折射率敏感的形式，如三次谐波产生 显像在这项工作中，我们假设三维时间分辨超分辨膜 成像将提供囊泡形成概况的直接读数， 和力量在这些研究中，我们将发展超分辨率多光子显微镜来监测MSC 成骨分化过程中胶原蛋白的产生和矿化囊泡的释放。然后我们将应用这个 活体超分辨三次谐波自适应散射波前校正技术 代显微镜监测MSC基质囊泡的形成和产生高效能的成骨细胞。 这将从技术的角度大大推进科学的理解，使标签- 通过完全不透明的方式实现纳米尺寸特征的免费超分辨率三维可视化 高度分散的骨组织我们有可能改变骨骼疾病的治疗，结果是， 将广泛适用于一系列人类健康问题。

PROJECT SUMMARY Mesenchymal stem cells (otherwise known as mesenchymal stromal cells or MSCs) hold great promise for treatment of bone related disorders due to their paracrine, multi-potential and immunosuppressive effects. One major objective is to leverage the osteogenic differentiation of MSCs as potential treatments in metabolic bone disorders (e.g. osteoporosis, osteogenesis imperfecta, or hypophosphatasia) or localized bone defects. However, knowledge of the MSC differentiation control pathways and how to leverage these during bioprocessing is lacking and has impeded more widespread clinical translation1-3. Unfortunately, the current clinical and industry state-of-the-art for characterization of MSC potency is to simply measure a few surface markers using flow cytometry and to study the ability of cells to differentiate in vitro into the target tissues (e.g. bone/cartilage). These assays are challenging to quantify, slow to perform, and have low direct relevance to in vivo functioning4. Our proposal aims to leverage development of novel imaging technologies to identify in vivo processes of key significance to transplanted stem cell differentiation and healthy bone tissue production, and will interrogate and leverage membrane lipid pathways to maintain and enhance the differentiation potential of MSCs. Our planned research is motivated by growing appreciation of how lipid membrane processing regulates cellular properties of central regulatory importance in bone formation, where maturing osteoblasts produce and secrete mineralizing matrix vesicles in multivesicular bodies of ~500 nanometer diameter that nucleate the deposition and growth of hydroxyapatite mineral crystals in bone collagen5,6. Little is known about processes that drive the formation and secretion of these vesicles, which is a pathway of central importance in the formation of mature cartilage and bone. This formation process is challenging to label, but with a high refractive index change it is ideal for refractive index sensitive modalities like third harmonic generation imaging. In this work, we hypothesize that 3-dimensional time-resolved super resolution membrane imaging will provide a direct readout of vesicle formation profiles that will predict cell differentiation and potency. In these studies, we will develop ultra-resolution multiphoton microscopy to monitor MSC collagen production and mineralizing vesicle release during osteogenic differentiation. We will then apply this technology in vivo using adaptive scattering wavefront correction for intravital super-resolution third harmonic generation microscopy to monitor MSC matrix vesicle formation and produce high potency osteogenic cells. This will significantly advance scientific understanding from a technological perspective by enabling the label- free super-resolution 3-dimensional visualization of nanometer-sized features through completely opaque highly scattering bone tissue. We have the potential to transform treatment of bone diseases, with results that would be broadly applicable to a range of human health concerns.