SMS microscopy

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 vast amount 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.

	Principle of SMS microscopy. Left: Only few markers in the sample reside in their bright state so that their diffraction-limited spots do not overlap on the detector. Their localized positions are registered in a histogram. Middle: After the markers have been read out and switched off in the first cycle, new markers are switched on and localized. Right: These cycles are repeated until sufficiently many positions have been registered to reconstruct the object.

Themes within SMS microscopy:

• 4Pi-SMS microscopy

Further information:

Hell, S. W. (2008):
"Microscopy and its focal switch"
Nature Meth. 6 (1), 24 - 32, Perspective, Special Feature, See Method of the year 2008

Egner, A., 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 (2007):
"Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters"
Biophys. J. 93, 3285 - 3290

Geisler, C., A. Schönle, C. von Middendorf, H. Bock, C. Eggeling, A. Egner, S.W. Hell (2007):
"Resolution of Lambda /10 in fluorescence microscopy using fast single molecule photo-switching"
Appl. Phys. A 88, 223-226