117 research outputs found
Quantum Dot Blueing and Blinking Enables Fluorescence Nanoscopy
No full textWe demonstrate superresolution fluorescence imaging of cells using bioconjugated CdSe/ZnS quantum dot markers. Fluorescence blueing of quantum dot cores facilitates separation of blinking markers residing closer than the diffraction barrier. The high number of successively emitted photons enables ground state depletion microscopy followed by individual marker return with a resolving power of the size of a single dot (similar to 12 nm). Nanoscale imaging is feasible with a simple webcam
4Pi microscopy with negligible sidelobes.
No full textThe coherent addition of the wavefronts of two opposing highangle lenses provides an axial (z) resolution improvement by 5 - 7-fold in farfield fluorescence microscopy. However, all microscopy concepts based on this principle have so far required mathematical deconvolution of the acquired data. This stems from the fact that the decrease of the axial width of the effective point spread function (EPSF) is accompanied by a substantial elevation of the side maxima of the EPSF along the optical axis. Here, we realize an EPSF with negligible lobes and gain axially superresolved images just through the physical phenomena involved. The constructive interference of the added wavefronts can be controlled through the image brightness which greatly simplifies the operation of the system
Molecular Orientation Affects Localization Accuracy in Superresolution Far-Field Fluorescence Microscopy
No full textWe investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-held superresolution microscopy based on stochastic single molecule switching, If tilt angle and defocus are unknown, the localization contains systematic errors up to about +/-125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about +/-32 nm, restricting the attainable resolution accordingly
STED nanoscopy with mass-produced laser diodes.
No full textWe show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing
2,2 '-thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy
No full textThe use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 mu m deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,2'-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures
Fluoreszenznanoskopie einzelner DNA-Moleküle mit Fluoreszenzverhinderung durch stimulierte Emission (STED)
No full textScharfe Aufnahmen: STED-Nanoskopie (STED=stimulierte Fluoreszenzverhinderung) von einzelnen DNA-Strängen visualisiert Strukturen mit einer 5- bis 6fach höheren Auflösung als konfokale Mikroskopie (Konf), wie aus dem linken bzw. rechten Bild ersichtlich ist. Die STED-Technik enthüllt Strukturen der Größenordnung der DNA-Persistenzlänge (ca. 50 nm), ohne dabei in maßgeblicher Weise Photoschäden wie Bleichen oder DNA-Bruch zu verursachen
Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots.
No full textUnlimited possibilities: Light-driven modulation of the fluorescence from Mn-doped ZnSe quantum nanocrystals has been established through excited-state absorption and its direct competition with spontaneous emission. Such optical control over electronic transitions enables far-field fluorescence microscopy analysis with diffraction-unlimited resolution (45 nm, red) based on quantum dots (confocal imaging has a resolution of 200 nm, blue)
STED nanoscopy with mass-produced laser diodes.
No full textWe show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing
Far-field optical nanoscopy with reduced number of state transition cycles.
No full textWe report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching
Tissue multicolor STED nanoscopy of presynaptic proteins in the calyx of held
No full textThe calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals
- …
