Fluorescence imaging flow cytometry in non-straight microfluidic channels

可以利用流动产生的力来操纵细胞，以探测其机械性能或将货物(例如药物)输送到细胞中。这可以在特殊的微流控装置中进行。由于高流速和复杂的几何结构，这些设备的一个限制是目前不可能在细胞流过时拍摄荧光图像。这些图像对于了解细胞在这些极端条件下的行为至关重要，因为荧光可以报告各种细胞成分(细胞骨架、膜、线粒体)对压力的反应。本项目的目的是实现一种高速荧光光片显微镜，在非直的微流体平台上观察高剪应力下的细胞。这将被用来提高我们对细胞对剪切变形的反应的理解，剪切变形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横流连接的几何结构，其中流体通过两个独立的通道流入和流出，细胞在固定的中心点经历较大的剪切应力。然而，这种几何形状使实现荧光显微镜具有挑战性。将使用倾斜平面显微镜，因为这项技术能够应用倒置几何形状的光片显微镜(从底部查看微流控设备)。与此同时，光片对于最大限度地减少散焦荧光以产生高速高对比度图像至关重要。在这种设置下，目标是使用增强型高速相机实现每秒20万帧的成像。这又将允许在流式细胞仪中以高达1米/S的速度流动的情况下测量100,000个细胞/分钟。当细胞承受高剪应力时，它们的反应是经历大量的拉伸。这种伸展已经被证明取决于细胞的类型，这样交叉流式细胞术就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导致变形知之甚少，这就是新的细胞仪将被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，以解释增加的表面积。这为药物输送或小分子进入细胞提供了潜在的机会，因为这些孔是在变形过程中产生的。随着荧光显微镜的加入，我们对这些毛孔产生的细胞机制的理解将得到改善。随着对孔形成(例如，孔大小分布、寿命、稳定性)的深入了解，有可能对它们作为细胞内输送载体的能力进行建模和描述。

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.