Video-Rate Super Resolution Microscopy in Living Cells

Super resolution microscopy based on single-molecule localization relies on precise and accurate localization of large numbers of single-molecules. However, the necessity of accumulating large numbers of localization estimates limits the time resolution typically to seconds to minutes1,2.Two major limitations are the acquisition speed and the photon budget. Replacing the usually used EMCCD with a recently introduced sCMOS camera results in leaps in both acquisition speed and effective quantum efficiency. However, the intrinsic pixel-dependent Gaussian noise of the sCMOS cameras introduces localization artifacts and greatly reduces the reliability of the results.Here, we present a set of specially designed methods that characterize an sCMOS camera for the first time and allow for unbiased and precise localization analysis. Using this method we demonstrate Cramer-Rao lower bound-limited single-molecule localization with an sCMOS camera. Combining the novel algorithm with a recently developed multi-emitter fitting algorithm3, We shortens the typical acquisition time for fixed samples by up to two orders of magnitude without compromising the field of view. Furthermore, we demonstrate localization-based super-resolution microscopy in live cells by monitoring dynamics of protein clusters, vesicles and organelles at a temporal resolution from 2 to 30 frames per second4.These methods allowed us to investigate cytokinetic apparatus in live fission yeast at 20-30 nm resolution. In general, the significantly improved temporal resolution allows super resolution imaging of a large range of dynamic events in living cells.1. Patterson, G., Davidson, M., Manley, S. & Lippincott-Schwartz, J. Annu. Rev. Phys. Chem.61, 345-67 (2010).2. Gould, T. J., Hess, S. T. & Bewersdorf, J. Annu. Rev. Biomed. Eng.14, 231-54 (2012).3. Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Biomed. Opt. Express 2, 1377-93 (2011).4. Huang, F. et al.Nat. Methods 10, 653-8 (2013).