24 February 2009 Photoswitching microscopy with subdiffraction-resolution
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Abstract
High-resolution fluorescence imaging has a vast impact on our understanding of intracellular organization. The key elements for high-resolution microscopy are reversibly photo-switchable fluorophores that can be cycled between a fluorescent and a non-fluorescent (dark) state and can be localized with nanometer accuracy. For example, it has been demonstrated that conventional cyanine dyes (Cy5, Alexa647) can serve as efficient photoswitchable fluorescent probes. We extended this principle for carbocyanines without the need of an activator fluorophore nearby, and named our approach direct stochastic optical reconstruction microscopy (dSTORM). Recently, we introduced a general approach for superresolution microscopy that uses commercial fluorescent probes as molecular photoswitches by generating long lived dark states such as triplet states or radical states. Importantly, this concept can be extended to a variety of conventional fluorophores, such as ATTO520, ATTO565, or ATTO655. The generation of non-fluorescent dark states as the underlying principle of superresolution microscopy is generalized under the term photoswitching microscopy, and unlocks a broad spectrum of organic fluorophores for multicolor application. Hereby, this method supplies subdiffraction-resolution of subcellular compartments and can serve as a tool for molecular quantification.
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Sebastian van de Linde, Sebastian van de Linde, Mark Schüttpelz, Mark Schüttpelz, Robert Kasper, Robert Kasper, Britta Seefeldt, Britta Seefeldt, Mike Heilemann, Mike Heilemann, Markus Sauer, Markus Sauer, } "Photoswitching microscopy with subdiffraction-resolution", Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 71850F (24 February 2009); doi: 10.1117/12.810261; https://doi.org/10.1117/12.810261
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