Robert Kasper
Bielefeld University
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Publication
Featured researches published by Robert Kasper.
Small | 2010
Robert Kasper; Benjamin Harke; Carsten Forthmann; Philip Tinnefeld; Stefan W. Hell; Markus Sauer
In recent years, fluorescencemicroscopy techniques have been invented that are no longer fundamentally limited by diffraction despite using visible light focused by conventional optical elements. Contrary to earlier attempts to improve the spatial resolution, such as near-field optics and aperture filters, all far-field fluorescence ‘‘nanoscopy’’ methods known to date rely on a judicious exploitation of selected fluorophore properties. In particular, all are based on utilizing a molecular mechanism that renders the fluorophores incapable of responding with fluorescence emission to excitation light. This fluorescence inhibition mechanism is implemented in the image formation in such away that fluorophores that are closer than the diffraction limit emit sequentially in time and hence can be discerned. In stimulated emission depletion (STED) microscopy, fluorescence is inhibited by subjecting the dye molecules to an additional beam of light, thus inducing stimulated emission from the fluorescent state S1 to the ground state S0. The
Journal of Biophotonics | 2008
Britta Seefeldt; Robert Kasper; Thorsten Seidel; Philip Tinnefeld; Karl-Josef Dietz; Mike Heilemann; Markus Sauer
We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications.
Single Molecule Spectroscopy and Imaging II | 2009
Sebastian van de Linde; Mark Schüttpelz; Robert Kasper; Britta Seefeldt; Mike Heilemann; Markus Sauer
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.
Proceedings of SPIE, the International Society for Optical Engineering | 2007
Daniel Fetting; Robert Kasper; Philip Tinnefeld
Most biological processes are governed by assemblies of several dynamically interacting molecules. We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. By comparing energy transfer of the different subpopulations, we can disentangle the reasons that lead to the occurrence of three-way junctions lacking one chromophore. A merit of the method is the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously.
Angewandte Chemie | 2008
Mike Heilemann; Sebastian van de Linde; Mark Schüttpelz; Robert Kasper; Britta Seefeldt; Anindita Mukherjee; Philip Tinnefeld; Markus Sauer
Angewandte Chemie | 2008
Jan Vogelsang; Robert Kasper; Christian Steinhauer; Britta Person; Mike Heilemann; Markus Sauer; Philip Tinnefeld
Journal of the American Chemical Society | 2005
Mike Heilemann; Emmanuel Margeat; Robert Kasper; Markus Sauer; Philip Tinnefeld
Applied Physics B | 2008
S. van de Linde; Robert Kasper; Mike Heilemann; Markus Sauer
Angewandte Chemie | 2008
Mike Heilemann; Sebastian van de Linde; Mark Schüttpelz; Robert Kasper; Britta Seefeldt; Anindita Mukherjee; Philip Tinnefeld; Markus Sauer
Journal of the American Chemical Society | 2006
Mike Heilemann; Robert Kasper; Philip Tinnefeld; Markus Sauer