Live cell imaging and fluorescence microscopy
At Umeå University, we have access to a wide range of high-end light microscopy instruments through the biochemical imaging centre Umeå (BICU). Epi- and confocal microscopy is commonly used by us to visualize infected cells and to localize fluorescent molecules or viral components in various cellular compartments e.g. via immunostaining or using fluorescent proteins. Furthermore, we use fluorescence recovery after photobleaching (FRAP) to study the diffusion of molecules within lipid membranes and total internal fluorescence (TIRF) microscopy to track individual particles as they interact with the membranes.
In our research, we are particularly interested in dynamic phenomena related to viral infection. To study such processes, we have access to a high-end epifluorescence microscope equipped with laser-based total internal reflection fluorescence (TIRF) illumination and fluorescence recovery after photobleaching (FRAP) capabilities is located directly in our lab.
In a FRAP experiment, a small area of a membrane containing fluorescent molecules is exposed to a high energy laser beam leading to localized bleaching of the fluorophores. By observing recovery of the fluorescent signal over time, one can therefore study the mobility of the fluorescently labelled molecules (Figure A). If the membranes are static, the bleaching spot will remain visible, if the lipids freely diffuse, the dark spot will fade out over time. We use FRAP to look at the diffusion of fluorescently labelled lipids and proteins within cellular membranes or cell-surface mimics.
TIRF microscopy is a surface sensitive microscopy technique which allows for the visualization of fluorescent objects found within ~100 nm from the surface of a glass substrate while discriminating them from the ones found in solution. We use TIRFM, both to look at the recruitment of viruses or intracellular organelles at the basal membrane of live cells, as well as to probe interaction kinetics, specifically virus attachment and detachment, as well as diffusion of individual virus particles interacting with cell surface mimics (read more) (Figure B). [1, 2]
Key References