Cellular mechanics of particle binding and phagocytosis investigated by photonic force microscopy and high-speed imaging

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The binding of particles such as bacteria, viruses or debris to living cells and the possible uptake into the cell interior – phagocytosis – represent central processes in cell biology and immunology. The observation of such binding and uptake processes, which often occur stepwise, is limited by the spatial and temporal resolution of light microscopes. Despite modern fluorescence techniques, fluctuation based processes, such as the (re-)organization of molecular bonds can hardly be observed or analyzed. Therefore, many partial processes like binding, particle transport or engulfment into the cell body are not sufficiently understood, especially not regarding the cell mechanics, since they occur on scales of nanometers and milliseconds, which are difficult to address by most optical methods.This proposal bases on the results of the first proposal and aims to extend the measurement and analysis methods developed therein. Using the established technique of Photonic Force Microscopy, interaction processes between particle and living cells shall be induced and then controlled variably in space and time. Binding, particle transport at the surface and the actual uptake consist of many multi-scale processes (10^-9 to 10^-5 meters and 10^-5 to 10^2 seconds) and can be better understood, if these processes can be measured on a large temporal and spatial bandwidth. This shall be accomplished by using fast, coherent, i.e. label-free methods such as interferometric 3D particle tracking (at 1 MHz sampling rate) or a novel live-cell super-resolution microscopy method, based on rotating coherent scattered light (ROCS, 100Hz sampling rate). On the other side, fluorescence-based confocal spinning disc scanning shall be extended by an electro-optical tunable lens to enable fast acquisition of 3D image stacks.In this proposal, two PhD students, PhD1 and PhD2, shall work on the following projects: using the above mentioned optical methods, PhD1 shall investigate the direct binding and uptake of particles at flat cell membranes of mouse macrophages. PhD1 shall research, when and how a cell can sense and respond to an approaching particle (e.g. by actin reorganization). Furthermore, PhD1 shall measure the stepwise engulfment by the cell membrane, and analyze the local change of the 3D binding stiffness and friction extracted from the particle’s fluctuations. PhD2, on the other side, shall investigate binding and uptake of the particle mediated by filopodia and lamellipodia. PhD2 shall analyze to what extent the retraction of filopodia can be self-optimized. In addition, the frequently observed, discontinuous transport of particles along filopodia and lamellipodia and the underlying mechanical coupling processes to the dynamic cytoskeleton shall be researched and analyzed.