STED microscopy—super-resolution bio-imaging utilizing a stimulated emission depletion
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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 2015Keywords:
STED microscopy
Two-photon excitation microscopy
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.
STED microscopy
Point spread function
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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.
STED microscopy
Point spread function
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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
STED microscopy
Two-photon excitation microscopy
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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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Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.
STED microscopy
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Stimulated emission, predicted by Albert Einstein in 1917, not only prepared the grounds for the invention of the laser, but also for a far-field fluorescence microscopy with diffraction-unlimited resolution. In stimulated emission depletion (STED) microscopy, stimulated emission is not used for light amplification but for a saturated quenching of the fluorescence emission. After demonstrating a five-fold improvement of the lateral (x and y) resolution over the diffraction barrier, we apply STED microscopy to nanostructures of stained PMMA. For the first time, periodic line structures of 80 nm width and 40 nm gaps are resolved with focused visible light.
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Light microscopy is a key scientific instrument in the life sciences. However, the resolution of far-field light microscopy is limited by diffraction. Exploiting a saturated depletion of the molecular excited state by stimulated emission, stimulated emission depletion (STED) breaks the resolution barrier in the important subfield of fluorescence microscopy. To this end, STED microscopy utilizes a doughnut-shaped beam featuring a central zero which is capable of quenching the fluorescence solely in the focal periphery. While STED microscopy was initially restricted to near infrared-emitting fluorophores, in this thesis STED microscopy is shown to be viable with visible (green-yellow-red) fluorophores. In particular, STED is established with the green and yellow fluorescent proteins (GFP, YFP) which are endogenous cellular markers of outstanding biological importance. The spectral conditions for STED with these fluorophores are given. The expansion of STED to the visible range has enabled the first application to biophysics and to addressing unsolved problems of cell biology. For example, STED microscopy revealed that the synaptic vesicle protein synaptotagmin remains an integral patch following fusion with the plasma membrane. Moreover, membrane microdomains were resolved with unprecedented (65 nm) spatial resolution. Furthermore, a novel doughnut-shape of the STED beam utilizing a helical phase ramp and a central singularity was established for STED. Finally, due to the smaller wavelength for stimulated emission, the viability of STED with visible dyes pushes the resolution down to smaller attainable values.
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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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Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.
STED microscopy
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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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