Fluorescence imaging flow cytometry in non-straight microfluidic channels

细胞可以利用流动产生的力来操纵，既可以探测它们的机械特性，也可以将货物（如药物）输送到细胞中。这可以在特殊的微流体装置中进行。由于高流速和复杂的几何结构，这些设备的局限性是，目前不可能在细胞流过时拍摄荧光图像。这些图像对于了解这些极端条件下的细胞行为至关重要，因为荧光可以报告各种细胞成分（细胞骨架、膜、线粒体）对应激的反应。本项目的目的是实现一种高速荧光片显微镜，以观察非直线微流控平台中高剪切应力下的细胞。这将用于提高我们对细胞对剪切变形的反应的理解，这在识别癌细胞以及将探针和药物输送到细胞中具有潜在的应用。为了产生剪切应力，流式细胞仪将被制造成交叉流连接几何形状，其中流体通过两个独立的通道进出，细胞在一个固定的中心点经历较大的剪切应力。然而，这种几何形状使得荧光显微镜的实现具有挑战性。斜平面显微镜将被使用，因为这项技术能够应用光片显微镜在一个倒置的几何形状（从底部观察微流体装置）。同时，光片对于最小化失焦荧光以产生高速高对比度图像至关重要。通过这种设置，目标是使用强化的高速相机实现每秒20万帧的成像。这反过来又允许测量100,000个细胞/分钟，细胞在流式细胞仪中以高达1m /s的速度流动。当细胞经历高剪切应力时，它们的反应是进行大量拉伸。这种拉伸已被证明依赖于细胞的类型，因此交叉流式细胞术可以用来识别癌细胞。然而，对于在这些条件下哪些细胞成分负责变形知之甚少，这就是新细胞仪将应用于研究各种细胞成分如何控制变形的地方。此外，当细胞被拉伸时，细胞膜上形成了气孔，以解释表面积的增加。这为药物输送或小分子进入细胞提供了潜在的机会，因为这些孔隙在变形过程中产生。随着荧光显微镜的加入，我们对这些毛孔形成的细胞机制的理解将得到改善。随着对孔隙形成（如孔径分布、寿命、稳定性）了解的增加，有可能建模和描述它们作为细胞内递送运输载体的能力。

Cells can be manipulated using forces generated by flow, either to probe their mechanical properties or to deliver cargo (e.g. drugs) into the cells. This can be performed in special microfluidic devices. A limitation of these devices, due to the high flow rates and complex geometry, is that it is currently impossible to take fluorescence images of the cells as they flow through. These images are essential for understanding cell behaviour under these extreme conditions, as fluorescence can report on the response of various cellular components (cytoskeleton, membrane, mitochondria) to stress. The aim of this project is to implement a high-speed fluorescence light sheet microscope to view cells under high shear stress in a non-straight microfluidic platform. This will be used to improve our understanding of cellular responses to shear deformation, which has potential application of identifying cancerous cells as well as delivering probes and drugs into cells. In order to create a shear stress, a flow cytometer will be manufactured in a cross-flow junction geometry, where fluid flows in and exits through two separate channels and the cells experience large shear stresses at a stationary central point. This geometry however makes implementing a fluorescence microscope challenging. Oblique plane microscopy will be used, as this technology is able to apply light sheet microscopy in an inverted geometry (viewing the microfluidic device from the bottom). Meanwhile the light-sheet is essential to minimise out-of-focus fluorescence to generate high-contrast images at high speeds. With this setup the aim is to achieve imaging at 200,000 frames-per-second, using an intensified high-speed camera. This will in turn allow measurements of 100,000 cells/min with the cells flowing at speeds of up to 1 m/s in the flow cytometer. When cells experience high shear stresses they respond by undergoing substantial stretching. This stretching has been shown to depend on the type of cell, such that cross-flow cytometry can be used to identify cancerous cells. However, little is known about which cellular components are responsible for deformation under these conditions and this is where the new cytometer will be applied to study how various cell component control the deformation. Furthermore, as cells are stretched pores are formed in the cell membrane to account for the increased surface area. This offers potential opportunities for drug delivery or the transport of small molecules into cells due to these pores being created during deformation. With the addition of fluorescence microscopy, our understanding of the cellular mechanisms responsible for the creation of these pores will be improved. With an increased understanding of pore formation (e.g. pore size distribution, lifetime, stability), there is a possibility to model and describe their capability of acting as transport vehicles for intracellular delivery.