Cellular processes are regulated by complex interactions of biomolecules. The spatiotemporal organization of these biomolecules such as their localization to intracellular organelles is critical for their function. With the breakthrough of optical single-molecule and super-resolution microscopy techniques such as Photoactivated Localization Microscopy (PALM), it became possible to study the spatio-temporal organization of biomolecules on a nanoscopic length scale below the optical diffraction limit of conventional microscopes. However, the long data acquisition time of PALM precludes high-resolution measurements in living cells due to the motion of intracellular structures. Here, I will present our novel developments of motion-corrected PALM in living cells that overcome these limitations.

          The application of mcPALM to dCas9-based chromatin imaging revealed that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Furthermore, we gained a deeper understanding of lipid droplet regulation by following fatty acid incorporation and changes in enzyme densities based on metabolic needs of cells. Motion-corrected PALM therefore improves the ability to analyze the mobility and the time-averaged nanoscopic features of intracellular structures with single molecule precision and can yield unprecedented insights across length and time scales.

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