Showing papers by "Sebastian van de Linde published in 2009"
400 citations
TL;DR: The potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with approximately 20 nm optical resolution is demonstrated.
Abstract: We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as β-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with ∼20 nm optical resolution.
122 citations
34 citations
TL;DR: A simple method is described that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope.
Abstract: We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.
34 citations
12 Feb 2009
TL;DR: Direct stochastic optical reconstruction microscopy (d STORM) as discussed by the authors uses commercial fluorescent probes as molecular photoswitches by generating long-lived dark states such as triplet states or radical states.
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 (d STORM). 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.
3 citations