Fluorescence imaging flow cytometry in non-straight microfluidic channels

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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.