Optimizing Fluorophores For Super-resolution Fluorescence STED Microscopy
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STED microscopy
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The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular-shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut-shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.
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Stimulated emission depletion (STED) microscopy exploits nonlinear saturable optical transition of fluorescent molecules, allowed to overcome Abbe's diffraction-limit and provides diffraction-unlimited resolution in far-field optical microscopy. We elaborate the mechanism of STED and the conditions of depletion. The formula of STED microcopy resolution is deduced through effective point spread function (E-PSF). The STED system resolution is mainly dominated by the quality of the fluorescence depletion patterns in the focal plane. The depletion pattern is mainly affected by STED beam intensity, polarization, phase plate, primary aberrations, STED pulse shape, pulse duration and delay time. In this paper, we found related models and simulate the relationship between the depletion patterns and the parameters, and put forward effective approach to enhance the system resolution.
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Journal Article STED microscopy—super-resolution bio-imaging utilizing a stimulated emission depletion Get access Kohei Otomo, Kohei Otomo 1Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita, Sapporo 001-0020, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Terumasa Hibi, Terumasa Hibi 1Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita, Sapporo 001-0020, Japan2Graduate School of Information Science and Technology, Hokkaido University, Kita 14 Nishi 9, Kita, Sapporo 060-0814, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Yuichi Kozawa, Yuichi Kozawa 3Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Tomomi Nemoto Tomomi Nemoto * 1Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita, Sapporo 001-0020, Japan2Graduate School of Information Science and Technology, Hokkaido University, Kita 14 Nishi 9, Kita, Sapporo 060-0814, Japan *To whom correspondence should be addressed. E-mail: tn@es.hokudai.ac.jp Search for other works by this author on: Oxford Academic PubMed Google Scholar Microscopy, Volume 64, Issue 4, August 2015, Pages 227–236, https://doi.org/10.1093/jmicro/dfv036 Published: 06 July 2015 Article history Received: 06 April 2015 Accepted: 08 June 2015 Published: 06 July 2015
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Abstract We present localization with stimulated emission depletion (LocSTED) microscopy, a combination of STED and single-molecule localization microscopy (SMLM). We use the simplest form of a STED microscope that is cost effective and synchronization free, comprising continuous wave (CW) lasers for both excitation and depletion. By utilizing the reversible blinking of fluorophores, single molecules of Alexa 555 are localized down to ~5 nm. Imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography shows that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm, substantially improving the classical resolution of a CW STED microscope of about 60 nm. LocSTED microscopy also allows estimating the total number of proteins attached on a single nanoanchor.
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Resolution describes the possibility to separate structural features in the imaging process. In far-field optical microscopes the resolution is limited by wavelength and numerical aperture. Stimulated Emission Depletion Microscopy (STED) is a method to resolve structures below the limits of optical resolution and is therefore attributed to super-resolution. STED uses a differential method of two different diffraction patterns, where one pattern excites and the second pattern de-excites fluorochromes. The residual excited area is controllable by intensity down to a theoretically unlimited resolution.
Among the major steps in the development of STED microscopy is the use of continuous wave lasers (CW-STED). A recent investigation on the time course of the fluorescence emission probability in CW-STED has revealed the benefit of using temporal gating of fluorescence detection (gSTED) to further improve the resolution of CW-STED and to reduce the STED laser intensity in the sample for a given resolution.
There are many practical benefits of STED microscopy. It works directly on standard fluorophores and fluorescent proteins such as Alexa 488, Oregon Green 488 and FITC, eYFP, Citrin, and eGFP. As a point scanning implementation, STED provides optical section capabilities like those in confocal microscopy to allow super-resolution imaging within tissue. The method produces details smaller than 50 nm in a direct optical implementation that produces super-resolution raw data, to which contrast enhancing processes such as deconvolution can be applied.
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Stimulated emission depletion (STED) nanoscopy is a promising super-resolution imaging technique for microstructure imaging; however, the performance of super-resolution techniques critically depends on the properties of the fluorophores (photostable fluorophores) used. In this study, a suitable probe for improving the resolution of STED nanoscopy was investigated. Quantum dots (QDs) typically exhibit good photobleaching resistance characteristics. In comparison with CdSe@ZnS QDs and CsPbBr3 QDs, Cd-free InP/ZnSeS QDs have a smaller size and exhibit an improved photobleaching resistance. Through imaging using InP/ZnSeS QDs, we achieved an ultrahigh resolution of 26.1 nm. Furthermore, we achieved a 31 nm resolution in cell experiments involving InP/ZnSeS QDs. These results indicate that Cd-free InP/ZnSeS QDs have significant potential for application in fluorescent probes for STED nanoscopy.
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Fluorophores useful for STimulated Emission Depletion (STED) spectroscopy must fulfill strict requirements on depletion efficiency and photostability. These parameters determine the effective resolution of STED imaging. Resolution is typically measured on 30–80 nm spheres heavily decorated with STED bright fluorophores, limiting the possibility to estimate the true resolution achievable on a specific dye. Here we show how single molecule STED microscopy provides an estimate of the fluorophore stimulated emission cross section and of its photostability under STED irradiation. Fluorescein, a green and a yellow mutant of GFP, are tested, and the results are discussed and compared to those obtained with Chromeo488-covered 80 nm spheres on a commercial continuous-wave STED microscope.
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We propose stimulated emission depletion (STED) structured illumination microscopy (SIM), which has structured excitation and structured STED light with the same grating vector. Numerical simulation shows the possibility of improving resolution and reducing background fluorescence.
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