A5018426212- Advanced Fluorescence Microscopy Techniques
- Cell Image Analysis Techniques
- Advanced Electron Microscopy Techniques and Applications
- Advanced biosensing and bioanalysis techniques
- Photoreceptor and optogenetics research
- Photochromic and Fluorescence Chemistry
- Photosynthetic Processes and Mechanisms
- Click Chemistry and Applications
- Plant Molecular Biology Research
- Microbial Natural Products and Biosynthesis
- Computational Drug Discovery Methods
- Plasmonic and Surface Plasmon Research
- Molecular Biology Techniques and Applications
- Biosensors and Analytical Detection
- Listeria monocytogenes in Food Safety
- Luminescence and Fluorescent Materials
- Nanoplatforms for cancer theranostics
- Spectroscopy Techniques in Biomedical and Chemical Research
- Ubiquitin and proteasome pathways
- Near-Field Optical Microscopy
- Spectroscopy and Chemometric Analyses
- Viral Infectious Diseases and Gene Expression in Insects
- Image Processing Techniques and Applications
- Advanced Nanomaterials in Catalysis
- Microfluidic and Capillary Electrophoresis Applications
Max Planck Institute for Multidisciplinary Sciences
2022
Fraunhofer Institute for Translational Medicine and Pharmacology
2022
Max Planck Institute for Biophysical Chemistry
2014-2021
Bielefeld University
2005-2011
Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants image sequences blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated sequential multiple depth positions. Here we introduce multiplexed scheme for simultaneous acquisition focal planes. Using 3D cross-cumulants, show that sampling can be...
Photoswitchable fluorophores─proteins and synthetic dyes─whose emission is reversibly switched on off upon illumination, are powerful probes for bioimaging, protein tracking, super-resolution microscopy. Compared to proteins, dyes smaller brighter, but their photostability the number of achievable switching cycles in aqueous solutions lower. Inspired by robust photoswitching system natural we designed a supramolecular based fluorescent diarylethene (DAE) cucurbit[7]uril (CB7) (denoted as...
Abstract Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy living cells have relied on the use reversibly switchable fluorescent proteins (RSFP) emitting in green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red‐emitting RSFP enabling spatial resolution up four times higher than diffraction barrier. By co‐expressing and red RSFPs demonstrate two‐color both for single (“donut”) beam scanning...
Abstract Subdiffraction super‐resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT on living green tissue. Live‐cell requires and utilizes comparatively low light doses intensities to overcome diffraction barrier. We generated a transgenic Arabidopsis thaliana line expressing reversibly switchable fluorescent protein rsEGFP2 fused...
The near infrared (NIR) optical window between the cutoff for hemoglobin absorption at 650 nm and onset of increased water 900 is an attractive, yet largely unexplored, spectral regime diffraction-unlimited super-resolution fluorescence microscopy (nanoscopy). We developed NIR fluorescent protein SNIFP, a bright photostable bacteriophytochrome, demonstrate its use as fusion tag in live-cell STED nanoscopy. further dual color red-confocal/NIR-STED imaging by co-expressing SNIFP with...
Abstract One of the key photophysical properties fluorescent proteins that is most difficult to measure quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it unknown many proposedly molecules sample are indeed (for example due incomplete maturation, or presence dark states). Here, we use plasmonic nanocavity-based method absolute yield values commonly used...
Abstract Diffraction‐unlimited far‐field super‐resolution fluorescence (nanoscopy) methods typically rely on transiently transferring fluorophores between two states, whereby this transfer is usually laid out as a switch. However, depending whether induced in spatially controlled manner using pattern of light (coordinate‐targeted) or stochastically single‐molecule basis, specific requirements the are imposed. Therefore, utilized just for one class only. In study we demonstrate that...
Abstract Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance novel applications. Here we establish a link between photoswitching contrast, one the key parameters that dictate achievable resolution in nanoscopy applications, chromophore conformation non‐fluorescent state rsEGFP2, widely employed label REversible Saturable OpticaL Fluorescence Transitions...
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a on- and nonfluorescent off-state by illumination with light of different wavelengths. Negative switching RSFPs are switched from the to same wavelength that also excites fluorescence. Positive have reversed response, where fluorescence excitation induces transition off- on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these states achieve...
The subcellular localisation of proteins in intact living cells is an important means for gaining information about protein functions. Even dynamic processes can be captured, which barely predicted based on amino acid sequences. Besides increasing our knowledge intracellular processes, this facilitates the development innovative therapies and new diagnostic methods. In order to perform such a localisation, under analysis are usually fused with fluorescent protein. So, they observed by...
In order to localise tagged proteins in living cells, the surrounding cells must be recognised first. Based on previous work regarding cell recognition bright-field images, we propose an approach automated of unstained live Drosophila which are high biological relevance. achieve this goal, original methods were extended enable additional application alternative microscopy technique, since exclusive usage images does not allow for accurate segmentation considered cells. cope with increased...
Abstract Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance novel applications. Here we establish a link between photoswitching contrast, key parameter that largely dictates the achievable resolution in nanoscopy applications, chromophore conformation non-fluorescent state rsEGFP2, widely employed label REversible Saturable OpticaL Fluorescence Transitions...
Abstract Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a on- and non-fluorescent off-state in response to irradiation with light of different wavelengths. Negative switching RSFPs are switched from the same wavelength which also excites fluorescence. Positive have reversed where fluorescence excitation induces transition off- on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these states achieve...
The Cover Feature displays a comparison of chromophore conformations in the X-ray crystal structures reversibly photoswitchable fluorescent protein rsEGFP2 its on state (green) and V151L (purple) V151A (yellow) variants their off states. latter an increased photoswitching contrast, as indicated by displayed switching kinetics. design Virgile ADAM. More information can be found Research Article Dominique Bourgeois, Martin Weik co-workers.