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Visualizing the Actin and Microtubule Cytoskeletons at - JoVE

Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an 2012-08-15 · Imaging and physiology data were acquired using data acquisition boards (National Instruments, Austin, TX) and modiﬁed versions of the software ScanImage (14) with code written in MATLAB (The MathWorks, Natick, MA). STED laser powers were measured at the output of the microscope objective. Live neuron imaging was performed with a time-averaged Christian Eggeling Group in Jena Biophysical Imaging Lab - Super-resolution microscopy and membrane biophysics. STED, MINFLUX, FCS, iSCAT, SIM, SMLM, STED-FCS. Professor Prof.

Sted imaging

2021-03-15 · Cell imaging. The confocal and STED images were recorded using a commercial Abberior Instruments Expert Line microscope equipped with a 775 nm 40 MHz STED laser and a 640 nm excitation laser after 2020-04-07 · 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. Stimulated emission depletion (STED) nanoscopy is a promising fluorescence microscopy to detect unresolvable structures at the nanoscale level and then achieve a superior imaging resolution in materials science and biological research. However, in addition to the optimization of the microscope, luminescent m With the STED technique, however, the necessary resolution can be reached, and therefore, this type of positive identification may be possible (see Fig. 2e). To improve the resolution of STED imaging even further, increased laser depletion intensity and/or smaller fluorescent probe complexes might be useful.

Unfortunately, this increased optical resolution also leads to a drawback: because many fluorophores are depleted by the depletion laser, this also results in a lower signal (fewer photons) being captured by the detector. 2019-09-30 · First successful STED imaging of DNA in vitro was demonstrated by Persson et al by employing the standard DNA bis-intercalating dye YOYO-1 (figure 2(a) and figure 3(a)), which belongs to dimeric cyanine class of dyes. YOYO-1 shows a 500-fold fluorescence enhancement upon binding to DNA. STED instruments utilize a raster-scan imaging scenario similar to a laser-scanning confocal microscope.

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During STED imaging, samples are irradiated with strong light at a wavelength of 592 nm, 660 nm, or 775 nm. It is of crucial importance that the sample is not absorbing light at these wavelengths. Step 2: Choice of fluorophores There is a wide range of fluorophores performing well with STED imaging and experiments with low labeling densities designed to stay close to physiological conditions are typical. At the same time, the dead-time compensation of DynamicPLUS allows crisp images of high-signal samples, such as bright confocal images.

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To enhance contrast, raw 2P-STED image stacks were subjected to deconvolution (Huygens HuCore version 17.4, Scientific Volume Imaging b.v., Hilversum, The Netherlands) utilizing a theoretical PSF based on microscope parameters and classic maximum likelihood estimation (cmle) with a quality stop criterion of 0.01, automatic background estimation and a signal-to-noise ratio (SNR) of 15. 2021-03-15 · Cell imaging. The confocal and STED images were recorded using a commercial Abberior Instruments Expert Line microscope equipped with a 775 nm 40 MHz STED laser and a 640 nm excitation laser after 2020-04-07 · 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. Stimulated emission depletion (STED) nanoscopy is a promising fluorescence microscopy to detect unresolvable structures at the nanoscale level and then achieve a superior imaging resolution in materials science and biological research.

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.
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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. Imaging dendritic spines in the hippocampus in vivo is challenging because of its remote location more than 1 mm below the surface of the mouse brain. A pioneering study accomplished this with two-photon (2P) microscopy, but only over a period of a few hours (Mizrahi et al., 2004). Super-resolution imaging using the principles of stimulated emission depletion (STED) microscopy requires collinear excitation of a sample with a Gaussian-shaped excitation beam and a donut-shaped depletion beam whose spin (polarization) and orbital angular momentum (OAM) signs are aligned. STED is a fluorescence microscopy technique which uses a combination of light pulses to induce fluorescence in a small sub-population of fluorescent molecules in a sample.

For single color STED imaging, SiR-tubulin or SiR-actin were added to DIV15 neurons in the BrainPhys medium at a final concentration of 1 µM during 1.5 hr at 37°C. For dual color imaging, SiR-tubulin and SiR700-actin were added to to the BrainPhys medium at 1 µM final concentration each and neurons were exposed during 1.5 hr at 37°C.
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It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. Stimulated Emission Depletion Microscopy (STED) Imaging below the optical diffraction limit Stimulated emission depletion microscopy (STED) is a fluorescence microscopy technique that overcomes the diffraction limited resolution of confocal microscopes.

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With the Multicolour STED imaging with a single STED laser is also possible; and the Download scientific diagram | Super-resolution stimulated emission depletion ( STED) imaging in optically cleared kidney samples. All samples were stained for 9 Apr 2016 Resolution of STED microscopy is higher than confocal microscopy. All cells were infected with HSV-1 17+ strain for 6 h, then prepared for FISH 30 Mar 2016 FP7,brain STED,FP7-PEOPLE-2011-IEF,MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV(DE) 19 Jun 2012 In contrast to any currently used super resolution techniques, STED microscopy possesses a unique set of advantages, such as, rapid image 14 Oct 2015 Abstract: Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method beating the 28 Feb 2012 achieve using diffraction-limited microscopy. With recent ad- vancements like spectral multiplexing or live-cell imaging,.

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Now during image acquisition, the In stimulated emission depletion (STED) imaging, the excitation and depletion laser beams extend well beyond the focal plane in the imaged sample. av H Blom · 2017 · Citerat av 139 — Despite its short history, diffraction-unlimited fluorescence microscopy techniques We outline the prerequisites for successful STED imaging experiments, Vi presenterar ett protokoll för att använda STED mikroskopi samtidigt bilden High-resolution Time-lapse Imaging and Automated Analysis of Här presenterar vi ett detaljerat protokoll för imaging de aktin och mikrotubuli cytoskeletons på immun synapsen använder STED mikroskopi. In this chapter, we introduce several super-resolution imaging technologies for brain and synapses, including optical microscopy (STED, STORM), expansion Nanoscopy has revolutionized the study of subcellular architecture and dynamics and is on its way to becoming the new gold standard in fluorescence imaging With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and Weuse this procedure to demonstrate four color STED imaging of platelets with ≤40 multicolor, super resolution microscopy, photobleaching, STED, platelets, av V Kumra · 2018 — Site localisation on bacterial surface proteins using super resolution imaging.

775nm (~1ns) pulsed depletion. SLM 2020-12-08 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.