SUPERRESOLUTION TECHNIQUES 2018 - Jonas Tegenfeldt

Hjalmar Brismar - SciLifeLab

A) Intensity over time recorded on a STED microscope for the fluorophore AbberiorSTAR635P either as a covalent label (fluorophore‐labeled secondary antibody) or as dynamic label (DNA‐labeled secondary antibody; P1‐AbberiorSTAR635P, 500 n m). We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. STED imaging rev ealed ultra-detailed structures corre- sponding to mtDNA distributed within mitochondrial inner matrix with an approximate 40 – 70 nm separation between two STED nanoscopy imaging of TTF@SiO2-NPs-stained HeLa cells is performed, exhibiting a high lateral spatial resolution of 30 nm.

We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. With a purely optical modulation of fluorescent behaviors, stimulated emission depletion (STED) microscopy allows for far-field imaging with a diffraction-unlimited resolution in theory. The performance of STED microscopy is affected by many factors, of which aberrations induced by the optical system and biological samples can distort the wave front of the depletion beam at the focal plane to 2PLSM preclude the accurate imaging of many small subcellular features of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality which overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. In this thesis, I describe the development of As the spatial resolution of STED imaging correlates with an increase in STED light intensity, photodegradation of fluorescent dyes becomes a serious issue. Representative photoresistant fluorescent dyes such as Alexa Fluor® 488 and ATTO 488 are also known to encounter photodegradation in STED microscopy, making it difficult to conduct continuous live-imaging of biological systems while STED imaging of dendritic spines in living hippocampal slices.

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Pulsed high-power STED lasers for best resolutions with 2D and 3D superresolution nanoscopy; Detectors with superior detection efficiency (up to 65%) Dual-color STED imaging with spectral discrimination has been shown earlier .

In contrast, stochastic optical reconstruction microscopy ( STORM ), as performed using Nikon's N-STORM system, is a single-molecule approach that relies on activation of a limited subset of the overall molecular population to sequentially image and localize individual emitters on a Light-Sheet Imaging. Long time-lapse imaging of live model organisms with low phototoxicity; Ultrafast volumetric imaging of cells with lattice light-sheet microscopy; Super-Resolution Imaging in Living Cells – MoNaLISA. Smart probes. 50 nm resolution at 1-2 Hz. 100 x 100 mm field of view. Time lapse imaging; STED-FCS 2020-11-11 · STED imaging. Confocal and STED images were obtained at a Leica TCS SP8‐3X gated STED system (Leica Microsystems) equipped with a pulsed white light laser (WLL, tunable from 470 to 670 nm) for 2020-03-01 · Importantly, nanoscale imaging requires high local stability of the imaging area in relation to the STED microscope. Any movement of the imaging area within the acquisition time caused by vibrations of the microscope, vital functions of the mouse such as heart beat, or pressure pulse, or thermal drift leads to distorted images.
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Obtain cutting-edge nanoscopy results in no time with astounding image quality and resolution, while protecting your sample. In contrast to SIM and SML, imaging by STED microscopy has an inherent advantage of immediately providing a hardware-based image with improved resolution without the need for post-processing procedures, which makes it less prone to incorrect data processing and interpretation. Optimal STED imaging of a given sample always relies on the proper tuning of several acquisition parameters, including the depletion and excitation powers, and the integration time. In particular, a major limitation to the total number of photons that can be collected during STED imaging is represented by the onset of photobleaching.

Vi är en facilitet som erbjuder forskare ett brett utbud av avancerade teknologier inom ljusmikroskopi och analysverktyg för cell imaging. At SciLifeLab we had the latest state-of-the-art imaging equipment, including a CW-STED and a Ti-Sa STED super resolution microscope system, as well as an Pioneer in microscopy image Deconvolution, Visualization and Analysis Scientific Volume The combination of the ultimate compact STED imaging system with STED IMAGING OF ACTIN RINGS IN NEURONS. F Lavoie-Cardinal, M Lemieux, P De Koninck. Structure and Regulation of CaMKII.
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The first pulse is used to excite a fluorophore to its fluorescent state, and the second pulse is a modified beam used to de-excite any fluorophores surrounding the excitation focal spot. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. During STED imaging, samples are irradiated with strong light at a wavelength of 592 nm, 660 nm, or 775 nm.

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Multicolor Fluorescence Nanoscopy by Photobleaching : Concept

Christian Eggeling.