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Super-resolution microscopy by TIRF-SIM

Background and Problem: Super-resolution microscopy techniques require significantly more illumination photons than conventional micros-copies. This leads to either longer acquisition times and/or enhanced fluorescence bleaching making them difficult to apply to dynamic processes in biology.
Approach: Using a combination of structured illumination microscopy (SIM) and total internal reflection fluorescence (TIRF) we are able to achieve nearly 100 nm resolution inside living cells at a frame rate of nearly 10 Hz and more than 70 acquisitions without significant bleaching. Thereby we were able to investigate and understand the multi-motor transport of the cytoskeleton filament MreB inside living bacteria (B. Subtilis).

PvO-yeast-Sag1 - TIRF.png PvO-yeast-Sag1 - TIRF-SIM.png     originalMreB_alive_01_brightfield-2.png originalMreB_alive_stack01_deltaT10s_driftCorr-1.gif
Yeast cell with the fluorescently labeled membrane protein Sag1. (Left) Normal TIRF image. (Right) TIRF-SIM image of the same cell showing higer resolution and contrast.  Scale bar is 1 µm. (Collaboration with the lab of R. Wedlich-Söldner)
MreB dynamics in the bacteria Bacillus Subtilis. (Left) Brightfield image of a B. subtilis cell. (Right) TIRF-SIM time series of GFP-labeled MreB in the same cell showing high dynamics. Scale bar is 1 µm. (Collaboration with the lab of P. Graumann)
  TIRFSIM-TIRF Comparison HeLa cells
Time-lapse image comparison of a lifeAct-mCherry stained HeLa cell. Left: TIRF-SIM, right: TIRF.



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