Unveiling the dynamic nature of cellular nanopores in living endothelial cells by video-rate super-resolution microscopy

Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fastest and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high and the physical size of the instruments is still prohibitively large. Here, I will present our latest efforts towards realizing a new generation of compact, cost-efficient and high-speed SR-SIM instruments. Tight integration of the structured illumination microscope capable of video-rate image acquisition with instant image reconstruction enables us to realize a super-resolving fluorescence microscope with the look-and-feel of regular wide-field microscopy. I will demonstrate this by presenting dynamics of intracellular transport and movement in living cells, in particular the dynamics of liver cell fenestrations. These nano-sized pores in liver endothelial cells play a particularly crucial role in human physiology, which is reduced or lost during disease and/or aging. To best address these issues from all perspectives, we utilize a suite of multimodal methods, e.g. the combination of optical tweezers with optical nanoscopy, or the combination of temporal and spatial methods of improving the spatial resolution and select the best possible method for each research question. I will present the pros and cons of these methods, their combination, and their applications on select biomedical examples.