CAREER: High-throughput Single-cell Biophysics

[150588]细胞的力学特性，细胞骨架和细胞内结构中这些特性的潜在起源，以及这种结构与疾病状态的关系，一直是一个迅速扩大的研究领域。纯粹的生物物理（机械）测量可以帮助揭示疾病和细胞结构之间的基本关联，这些特性已被证明是细胞表型的强大无标记生物标志物。例如，在细胞系和人体活检样本中，对力（可变形性）响应的形状变化的测量已被证明是恶性转化的指示。然而，以前的研究仅限于低通量分析技术的概念验证应用。在生物物理学研究、临床诊断或药物筛选中的应用必须需要大量的样本来获得具有统计意义的数据——从而产生敏感和特异性的结果。PI旨在解决当前测量技术的低通量和复杂性，使用下一代仪器在几分钟内检测数千个单细胞的机械特性，实现流式细胞术的易用性和通量。PI已经开始在拉伸（纯拉伸）流动中使用连续的微流体拉伸细胞来解决这一挑战。“可变形性血细胞计数”。PI已经证明了这种方法每秒变形1000个细胞的可行性，这比金标准机械测量技术高出几个数量级。为了扩展该仪器的开发，PI建议为细胞生物物理学和生物工程社区提供高通量和系统的数据，以帮助理解生物分子在细胞完整性中的作用，并最终为临床诊断应用提供目前未知的联系。智力水平细胞的力学性能被认为在很大程度上取决于具有最高刚度的细胞骨架元素（即肌动蛋白和微管）。近年来，中间丝、核膜蛋白和染色质结构被认为对细胞力学性能有重要影响。例如，已知多能干细胞和活化淋巴细胞缺乏核膜蛋白层蛋白A/C和较少凝聚的染色质。这些特征是否导致了这些细胞中观察到的高度可变形性仍然是一个悬而未决的问题。PI的创新之处在于研究这些元素在全细胞大范围变形中的组合，而不偏向于传统检查的细胞骨架中的优势起源。很明显，这些元素之间的相互作用可能控制某些细胞的机械行为，而单个元素可能主导其他细胞的反应（例如核质比大的淋巴细胞的染色质结构）。一个简单易用和高通量的仪器将允许对这些贡献者进行系统的调查，单独或作为潜在连接的机械网络的一部分，这将是生物工程和生物物理学团体试图解决细胞力学特性的基本起源的福音。更广泛的影响通过消除抗体标记的成本和减少与准备用于诊断的标记样品相关的技术人员劳动，无标记细胞状态的机械测量可以帮助解决医疗保健的高成本问题。此外，这种方法可以通过提高我们的诊断能力来提高生活质量，从而快速确定正确的治疗方法。教育和传播活动将与拟议的研究无缝结合。这些活动将集中在解决各种利益相关者的领域，包括(i)本科生和研究生社区，（ii）微流体社区，以及（iii）更广泛的公众。四项教育和传播活动包括：(1)与加州大学洛杉矶分校的CEED（卓越工程和多样性中心）合作，研究生和本科生接触高速相机操作和微流体。(2)夏季本科生实习，帮助经济困难的本科生参与研究。(3)一个分享和讨论微流控设计的在线社区。(4)在YouTube上建立一个“酷”的慢动作科学和说明性视频库（例如，装满不同粘度液体的水球被扔到墙上），以吸引和激发公众的兴趣。

1150588Di CarloThe mechanical properties of cells, the underlying origins of these properties in cytoskeletal & intracellular structure, and the relationship of this structure with disease states has been a rapidly expanding area of research. Purely biophysical (mechanical) measurements can help uncover fundamental associations between disease and cell architecture, and these properties have been shown to be powerful label-free biomarkers for cell phenotype. For example, a measure of shape changes in response to force (deformability) has been shown to be indicative of malignant transformation in cell lines and human biopsy samples. However, previous studies have been limited to proof-of-concept applications by the low-throughput analytical technologies employed. Applications in biophysics research, clinical diagnostics or drug screening necessarily require large sample sizes to obtain statistically significant data - leading to sensitive and specific results. The PI aims to address the low throughput and complexity of current measurement techniques using a next generation instrument to assay the mechanical properties of thousands of single cells in minutes achieving the ease-of-use and throughput of flow cytometry. The PI has started to address this challenge using continuous microfluidic stretching of cells in an extensional (purely stretching) flow - i.e. "Deformability Cytometry". The PI has demonstrated the feasibility of this approach for deforming 1000 cells per second, which is orders of magnitude higher than gold standard mechanical measurement techniques. Extending the development of this instrument, the PI proposes to provide high-throughput and systematic data to the cell biophysics and bioengineering communities that can aid in understanding the roles of biomolecules in cellular integrity, and ultimately suggest currently unknown connections to clinical diagnostic applications.Intellectual MeritThe mechanical properties of cells have been thought to be largely determined by cytoskeletal elements with the highest stiffness (namely actin and microtubules). Recently, intermediate filaments, nuclear envelope proteins and chromatin structure have been suggested to contribute significantly to cell mechanical properties. For example, pluripotent stem cells and activated lymphocytes are known to lack the nuclear membrane proteins lamin A/C and have less condensed chromatin. It remains an open question whether these characteristics are responsible for the high observed deformability in these cells. The PI's innovation is to investigate the combination of these elements on whole-cell large-scale deformation, without bias for dominant origins in the traditionally examined cytoskeleton. It is clear that the interplay between these elements may control mechanical behavior in some cells, while a sole element may dominate response in other cells (for example chromatin structure in lymphocytes with large nuclear to cytoplasmic ratios). A simple to use and high-throughput instrument will allow a systematic survey of these contributors, alone and as part of a potential linked mechanical network, which will be a boon to the bioengineering and biophysics communities attempting to address the fundamental origin of cell mechanical properties.Broader ImpactsA label-free mechanical measurement of cell state can help address the high cost of healthcare by eliminating the cost of antibody labels and reducing the technician labor associated with preparing labeled samples for diagnostics. Further, such an approach could improve quality of life by increasing our diagnostic capabilities - leading to the identification of the correct treatment quickly. Educational and dissemination activities will be seamlessly integrated with the proposed research. These activities will be focused in areas addressing a variety of stakeholders including the (i) undergraduate and graduate student community, (ii) the microfluidics community, and (iii) the broader public. The four education and dissemination activities include: (1) Graduate and undergraduate exposure to high-speed camera operation and microfluidics in collaboration with UCLA's CEED (Center for Excellence in Engineering and Diversity). (2) Summer undergraduate internships to assist financially disadvantaged undergraduates to become involved in research. (3) An online community for sharing and discussion of microfluidic designs. (4) An online YouTube gallery of "cool" slow-motion scientific and illustrative videos (e.g. water balloons filled with different viscosity fluids thrown against a wall) to engage and excite the public.