High resolution fluorescence microscopy uses the properties of fluorescent markers to overcome the diffraction limit. In contrast to ensemble-based methods (e.g. STED microscopy ), molecules are manipulated individually in SMS (single marker switching) microscopy. In this scheme, single markers are randomly selected from a large number of markers being in a dark state and transferred to a bright, detectable state by a stochastic on-switching process. The position of such a single molecule is calculated from its diffraction-limited fluorescence image which is separated spatially and temporally from the spots of other molecules. Here, the localization precision is better than the diffraction limit and scales with √N where N is the number of detected photons.
After the molecule is transferred to a dark state, this process of switching on, reading out and switching off of randomly selected markers is repeated a sufficient number of times; the histogram of all collected marker positions represents the final super-resolved SMS image.
Themes within SMS microscopy:
- S. W. Hell:
"Microscopy and its focal switch"
Nature Meth. 6 (1), 24 - 32, Perspective, Special Feature, See Method of the year 2008
- A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, S. W. Hell:
"Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters", Biophys. J. 93, 3285 - 3290, 2007
- C. Geisler, A. Schönle, C. von Middendorf, H. Bock, C. Eggeling, A. Egner, S. W. Hell:
"Resolution of Lambda /10 in fluorescence microscopy using fast single molecule photo-switching", Appl. Phys. A 88, 223-226, 2007
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