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Ground State Depletion Microscopy

Ground state depletion (GSD) microscopy is a RESOLFT technique that exhibits a time-sequential readout from within the diffraction zone at defined coordinates using reversible saturable or photoswitchable transitions. GSD requires lower laser intensities that STED or similar techniques because it employs the metastable triplet state, which has a lifetime range in the milliseconds to microseconds. GSD microscopy can be implemented in a manner similar to STED, as well as in single-molecule stochastic superresolution.

Hell, S. W.

Far-Field Optical Nanoscopy.  Science 316: 1153-1158 (2007).  An excellent overview of high-resolution fluorescence microscopy techniques that includes discussions of 4Pi, STED, GSD, RESOLFT, SPEM, PALM, and STORM. The author, whose own research in superresolution was instrumental in launching the field, also presents comparative images with the various methodologies.

Hell, S. W. and Kroug, M.

Ground-state-depletion fluorescence microscopy: A concept for breaking the diffraction resolution limit.  Applied Physics B: Lasers and Optics 60: 495-497 (1995).  Dr. Hell and Kroug introduce the concept of ground state depletion for overcoming the classical diffraction resolution limit. Although largely a theoretical paper, this effort indicates that lateral resolutions of less than 20 nanometers should be attainable with GSD microscopy.

Bretschneider, S., Eggeling, C. and Hell, S. W.

Breaking the diffraction barrier in fluorescence microscopy by optical shelving.  Physical Review Letters 98: 218103-4 (2007).  One of the original research reports by Dr. Stefan Hell's group on using the triplet state for superresolution imaging via ground state depletion (GSD). The authors note that GSD requires significantly less laser power than STED to achieve sub-diffraction resolution.

Folling, J., Bossi, M., Bock, H., Medda, R., Wurm, C. A., Hein, B., Jakobs, S., Eggeling, C. and Hell, S. W.

Fluorescence nanoscopy by ground-state depletion and single-molecule return.  Nature Methods 5: 943-945 (2008).  Application of the ground state depletion technique to single-molecule superresolution imaging using traditional fluorophores, such as rodamine and ATTO532. The technique monitors single emitters that return spontaneously from a dark state to the ground state.

Rittweger, E., Wildanger, D. and Hell, S. W.

Far-field fluorescence nanoscopy of diamond color centers by ground state depletion.  EPL Journal 86: 14001-6 (2009).  An elegant demonstration of optical shelving (driving fluorophores into a dark triplet state) using diamond color centers. The authors achieved a resolving power of approximately 7.6 nanometers, corresponding to 1/70 of the excitation wavelength.

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.

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.

van den Engh, G. and Farmer, C.

Photo-bleaching and photon saturation in flow cytometry.  Cytometry A 13: 669-677 (1992).  An early investigation into the effects of laser saturation on generating photobleaching and driving fluorophores into a dark state. The fluorophores investigated in terms of excitation-relaxation cycles were Hoechst nuclear dyes and propidium iodide.

Hackbarth, S. and Roder, B.

Anisotropy shape control via light quenching and ground state depletion.  Optics Communications 173: 247-254 (2000).  The authors compare the dependence of the fluorescence intensity versus excitation intensity and anisotropy for three different fluorophores while attempting to examine ground state depletion. It was determined that emission quenching and ground state depletion have a strong influence on the excitated state population.

Kolmakov K., Belov, V. N., Bierwagen, J., Ringemann, C., Mueller, V., Eggeling, C. and Hell, S. W.

Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy.  Chemistry: A European Journal 16: 158-166 (2010).  A report of novel rhodamine dyes that can be excited with a 630-nanometer laser and are extremely photostable with high fluorescent quantum yields and long excited state lifetimes. These fluorescent probes perform well using STED and GSD approaches.