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Reversible Saturable Optical Fluorescence Transitions (RESOLFT)

The term RESOLFT is a general concept that describes breaking the limiting diffraction barrier in far-field microscopy using reversible saturable or switchable optical transitions. The principle was first detailed in the form of stimulated emission depletion (STED) and ground state depletion (GSD) microscopy, where the diffraction barrier is broken by a saturated optical transition (depletion) between two states of a fluorescent probe. Although the concept of RESOLFT was originally developed using techniques that rely on point-spread function engineering, it is also useful to describe superresolution methodologies that rely on localization of single molecules.

Hell, S. W.

Fluorescence nanoscopy: Breaking the diffraction barrier by the RESOLFT concept.  NanoBiotechnology 1: 296-297 (2005).  An introduction to the concept of RESOLFT using STED and GSD as examples. Dr. Hell explains the theoretical concepts and equations underlying the concept and discusses limitations of the techniques.

Keller, J., Schonle, A. and Hell, S. W.

Efficient fluorescence inhibition patterns for RESOLFT microscopy.  Optics Express 15: 3361-3371 (2007).  The authors describe a method to effectively search for optimal zero intensity point patterns to derive a spatial intensity distribution that optimizes the focal plane resolution. This strategy is then expanded to apply under numerous experimental conditions.

Geisler, C., Schonle, A., von Middendorff, C., Bock, H., Eggeling, C., Egner, A. and Hell, S. W.

Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching.  Applied Physics A 88: 223-226 (2007).  A demonstration of nanoscale resolution (an order of magnitude beneath the diffraction limit) in far-field optical microscopy based on photoswitching of molecules according to the RESOLFT concept.

Schwentker, M. A., Bock, H., Hofmann, M., Jakobs, S., Bewersdorf, J., Eggeling, C. and Hell, S. W.

Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching.  Microscopy Research and Technique 70: 269-280 (2007).  Applying the RESOLFT concept to widefield imaging of switchable fluorescent proteins, the authors examine photoswitching in a red protein derived from the sea anemone Anemonia sulcata.

Bossi, M., Folling, J., Belov, V. N., Boyarskiy, V. P., Medda, R., Egner, A., Eggeling, C., Schonle, A. and Hell, S. W.

Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species.  Nano Letters 8: 2463-2468 (2008).  Using photoswitching between bright and dark states of optically bistable organic molecular probes, the authors demonstrate breaking the diffraction barrier with continuous wave irradiation.

Heintzmann, R., Jovin, T. M. and Cremer, C.

Saturated patterned excitation microscopy - A concept for optical resolution improvement.  Journal of the Optical Society of America A 19: 1599-1609 (2002).  An early theoretical implementation of the RESOLFT concept where nonlinear patterned excitation microscopy is used to achieve a substantial improvement in resolution by deliberate saturation of the fluorophore excited state.

Hofmann, M., Eggeling, C., Jakobs, S. and Hell, S. W.

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.  Proceedings of the National Academy of Sciences (USA) 102: 17565-17569 (2005).  The authors describe a technique for superresolution microscopy using fluorescent proteins that involves illumination intensities eight orders of magnitude smaller than those necessary for STED or GSD.

Hell, S. W.

Strategy for far-field optical imaging and writing without diffraction limit.  Physics Letters A 326: 140-145 (2004).  One of the first research reports outlining the theoretical aspects of non-linear optical imaging to break the diffraction barrier. A predicted subdiffraction resolution is formulated in a simple equation.

Enderlein, J.

Breaking the diffraction limit with dynamic saturation optical microscopy.  Applied Physics Letters 87: 094105-3(2005).  A theoretical paper that describes superresolution microscopy based on fast temporal measurements of the fluorescence decay in photoswitching probes after sudden switch-on of the light excitation.

Hell, S. W., Dyba, M. and Jakobs, S.

Concepts for nanoscale resolution in fluorescence microscopy.  Current Opinion in Neurobiology 14: 599-609 (2004).  An excellent review article on superresolution microscopy that discussed reversible saturable and photoswitchable optical transitions to effective achieve higher levels of resolution. Different approaches to RESOLFT microscopy are described.