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Dark States in Single-Molecule Superresolution Microscopy

The application of reversible photoswitching with traditional synthetic fluorophores and fluorescent proteins is promising to become a useful technique for superresolution imaging in cell biology. Due to the fact that most of these techniques rely on the transfer of fluorophores to a non-fluorescent dark state followed by precise localization of separated emitters, the photophysical properties of these fluorophores must be carefully controlled. The achievable resolution (and the ability to resolve structural features) depends on the brightness and labeling (molecular) density of the fluorophores, as well as the stability or lifetime of the non-fluorescent dark state.

Foelling, J., Bossi, M., Hannes, B., 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).  Report of a new technique known as ground state depletion with single-molecule return (GSDIM) that achieves superresolution based on switching traditional fluorophores to a metastable dark state and monitoring the position of those that spontaneously return to the ground state.

Heilemann, M., van de Linde, S., Schuettpelz, M., Kasper, R., Seefeldt, B., Mukherjee, A., Tinnefeld, P. and Sauer, M.

Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.  Angewandte Chemie International Edition 47: 6172-6176 (2008).  The original research report on direct stochastic optical reconstruction microscopy (dSTORM) that utilizes carbocyanine and Alexa Fluor dyes to achieve single-molecule superresolution imaging based on metastable dark states.

Baddeley, D., Jayasinghe, I. D., Cremer, C., Cannell, M. B. and Soeller, C.

Light-induced dark states of organic fluochromes enable 30 nm resolution imaging in standard media.  Biophysical Journal: Biophysical Letters 96: L22-L24 (2009).  Introduction of a technique known as reversible photobleaching microscopy (RPM) that employs single-molecule imaging to monitor the stochastic return of emitters from a dark state. Specimens were bathed in standard aqueous or glycerol based media to generate images having approximately 30-nanometer lateral resolution.

Steinhauer, C., Forthmann, C., Vogelsang, J. and Tinnefeld, P.

Superresolution microscopy on the basis of engineered dark states.  Journal of the American Chemical Society 130: 16840-16841 (2008).  The authors control fluorophore blinking for superresolution microscopy in standard saline solution after enzymatic removal of oxygen and addition of ascorbic acid. Using carbocyanine dyes and total internal reflection, blink microscopy is able to achieve a resolution of less an 50 nanometers.

Vogelsang, J., Cordes, T., Forthmann, C., Steinhauer, C. and Tinnefeld, P.

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy.  Proceedings of the National Academy of Sciences (USA) 106: 8107-8112 (2009).  An excellent demonstration of how adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent probes (such as ATTO 655) as efficient single-molecule photoswitches that can be imaged using superresolution microscopy techniques.

van de Linde, S., Kasper, R., Heilemann, M. and Sauer, M.

Photoswitching microscopy with standard fluorophores.  Applied Physics B 93: 725-731 (2008).  A detailed discussion of subdiffraction resolution fluorescence imaging based on light-induced photoswitching of individual fluorophores. In the presence of low concentrations of reducing, thiol-containing compounds, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores at approximately 20-nanometer resolution.

Lemmer, P., Gunkel, M., Weiland, Y., Mueller, P., Baddeley, D., Kaufmann, R., Urich, A., Eipel, H., Amberger, R., Hausmann, M. and Cremer, C.

Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range.  Journal of Microscopy 235: 163-171 (2009).  Using fluorescent proteins and synthetic dyes, the authors apply spectral precision distance microscopy (SPDM) to surpass the conventional resolution limits in widefield fluorescence microscopy using single-molecule techniques. The method is based on excitation intensity-dependent reversible photobleaching.

Heilemann, M., van de Linde, S., Mukherjee, A. and Sauer, M.

Super-resolution imaging with small organic fluorophores.  Angewandte Chemie International Edition 48: 6903-6908 (2009).  The authors present an impressive demonstration of single-molecule superresolution imaging using common Alexa Fluor and ATTO dyes. The synthetic probes are photoswitched using reducing thiol reagents and moderate laser intensity to control dark state properties and stochastic return to the ground state.

Vogelsang, J., Kasper, R., Steinhauer, C. Person, B., Heilemann, M., Sauer, M. and Tinnefeld, P.

A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes.  Angewandte Chemie International Edition 47: 5465-5469 (2008).  A technique designed to minimize photobleaching and blinking based on the removal of oxygen and quenching of triplet and charge-separated states by electron-transfer reactions. The reducing and oxidizing system (ROXS) was utilized to conduct single-molecule fluorescence spectroscopy of labeled oligonucleotides.

van de Linde, S., Sauer, M. and Heilemann, M.

Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores.  Journal of Structural Biology 164: 250-254 (2008).  The authors demonstrate application of commercially available fluorescent probes (Alexa Fluor 647) as photoswitches in localization microscopy to study the spatial organization of proteins in the mitochondria inner membrane.