Papers by Andreas Schönle
Scientific reports, Jan 20, 2017
Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, t... more Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between 'on' and 'off' states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells for ~100 × 100 μm(2) field...
Appl Phys a Mat Sci Process, 2007
By circumventing the optical diffraction limit, super-resolved fluorescence microscopies enable t... more By circumventing the optical diffraction limit, super-resolved fluorescence microscopies enable the study of larger cellular structures and molecular assemblies. However, fluorescence nanoscopy currently lacks the spatiotemporal resolution to resolve distances on the size of individual molecules and reveal the conformational fine structure and dynamics of molecular complexes. Here we establish FRET nanoscopy by combining colocalization STED microscopy with multiparameter FRET spectroscopy. We simultaneously localize donor and acceptor dyes of single FRET pairs with nanometer resolution and quantitatively measure intramolecular distances with sub-nanometer precision over a large dynamic range. While FRET provides isotropic 3D distance information, colocalization measures the projected distance onto the image plane. The combined information allows us to directly determine its 3D orientation using Pythagoras's theorem. Studying two DNA model systems and the human guanylate binding ...
Optics Express, 2012
In recent years, the diffraction barrier in fluorescence imaging has been broken and optical nano... more In recent years, the diffraction barrier in fluorescence imaging has been broken and optical nanoscopes now routinely image with resolutions of down to 20 nm, an improvement of more than 10 fold. Because this allows imaging much smaller features and because all superresolution approaches trade off speed for spatial resolution, mechanical instabilities of the microscopes become a limiting factor. Here, we propose a fully data-driven statistical registration method for drift detection and drift correction for single marker switching (SMS) imaging schemes, including a guideline for parameter choice and quality checks of the drift analysis. The necessary assumptions about the drift are minimal, allowing a model-free approach, but more specific models can easily be integrated. We determine the resulting performance on standard SMS measurements and show that the drift determination can be routinely brought to the range of precision achievable by fiducial marker-tracking methods.
Microscopy research and technique, 2007
We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high ... more We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.
Springer Handbook of Microscopy, 2019
CLEO:2011 - Laser Applications to Photonic Applications, 2011
ABSTRACT By transiently confining adjacent emitters to different states the diffraction limit in ... more ABSTRACT By transiently confining adjacent emitters to different states the diffraction limit in light microscopy can be overcome. We will discuss this fundamental concept underlying all current far-field nanoscopy approaches and present recent applications.
Nano letters, 2008
By combining the photoswitching and localization of individual fluorophores with spectroscopy on ... more By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.
Nature biotechnology, 2008
Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluoresc... more Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we d...
Nature methods, 2011
We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorop... more We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.
Biophysical Journal, 2008
Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly s... more Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at ;610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the onto off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.
Biophysical Journal, 2007
Applied Physics B, 2007
Rapid communication Lasers and Optics Applied Physics B h. bock c. geisler c.a. wurm c. von midde... more Rapid communication Lasers and Optics Applied Physics B h. bock c. geisler c.a. wurm c. von middendorff s. jakobs a. schönle a. egner s.w. hell u c. eggeling u Two-color far-field fluorescence nanoscopy based on photoswitchable emitters
Zum raumlich hochauflosenden Abbilden einer interessierenden Struktur einer Probe (2) wird eine S... more Zum raumlich hochauflosenden Abbilden einer interessierenden Struktur einer Probe (2) wird eine Substanz aus einer Gruppe von nicht schaltbaren Substanzen ausgewahlt, die einen ersten fluoreszenten elektronischen Zustand und einen zweiten nicht oder anders fluoreszenten elektronischen Zustand aufweisen; die aus ihrem ersten Zustand mit Licht (3), das sie zur Fluoreszenz anregt, anteilig in ihren zweiten Zustand uberfuhrbar sind und die spontan und/oder durch Einwirkung des Lichts (3) aus ihrem zweiten Zustand in ihren ersten Zustand zuruckkehren; wird die interessierende Struktur der Probe (2) mit der Substanz markiert; wird die Probe (2) auf ein Sensorarray (11) abgebildet, wobei eine raumliche Auflosungsgrenze der Abbildung groser (also schlechter) ist als ein mittlerer Abstand zwischen nachst benachbarten Molekulen der Substanz in der Probe (2); wird die Probe (2) in einem Bereich, der grosere Abmessungen als die raumliche Auflosungsgrenze aufweist, mit dem Licht (3) beaufschlagt...
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Papers by Andreas Schönle