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Examples of image blurring
Published in Mario Bertero, Patrizia Boccacci, Christine De Mol, Introduction to Inverse Problems in Imaging, 2021
Mario Bertero, Patrizia Boccacci, Christine De Mol
An extraordinary improvement of the resolution of CSLM is based on a technique proposed by Hell and Wichmann in 1994 [153] and experimentally demonstrated by Hell and Klar in 1999 [181]. For this basic invention and its development Stefan Hell was awarded the Nobel prize in Chemistry in 2014. This technique is now known as STimulated Emission Depletion (STED) microscopy and is able to bypass the diffraction limit of light microscopy (for a short review, see [296]). The most typical STED microscope uses a pair of synchronized laser pulses. The first excites the fluorophores of the specimen as in a conventional CSLM and produces an ordinary diffraction-limited focal point. The excitation pulse is followed by a red-shifted pulse which can desactivate the excited molecules before spontaneous emission. Therefore, in a suitable selected spatial region, the spontaneous decay can be anticipated by a decay stimulated by the second laser; this emitted light is suppressed by a suitable filter so that the image is formed by the light coming from the smaller region where spontaneous emission is occurring. It has been demonstrated that, by means of this technique, a resolution of few nanometers is possible. A comparison of CSLM image with the STED image of the same specimen is shown in Figure 2.11. The improvement in resolution is already evident by comparing the panels (a) and (b); it is even more evident by comparing the panels (c) and (d) which provide a zoom over the white squares indicated in the previous panels.
Super Resolution Microscopy
Published in John Girkin, A Practical Guide to Optical Microscopy, 2019
Stimulated emission and depletion (STED) microscopy is a beam scanned technique which requires very accurate timing of ultra-short pulses of laser light, high-speed detectors and suitable samples. The method was first described in 1994 (Hell and Wichmann 1994) and then reported as a practical system five years later (Klar and Hell 1999). The process uses one laser beam to excite a fluorophore and then a second beam, a very short while afterwards, with a “doughnut” intensity. This second beam removes the fluorescence from the molecules except in the centre of the doughnut beam which is black. The second beam is then switched off and the detector activated to collect the fluorescence from the remaining fluorescent molecules. As the centre of the doughnut beam has no light present it cannot diffract and hence can be made, at least in principle, arbitrarily small.
Basic Concepts of Laser Imaging
Published in Helmut H. Telle, Ángel González Ureña, Laser Spectroscopy and Laser Imaging, 2018
Helmut H. Telle, Ángel González Ureña
In summary, STED microscopy allows for superresolution imaging in the range of around 50 nm. However, this increase in optical resolution comes at a price. Because the majority of initially excited fluorophores are quenched by the STED-depletion laser, the result is a much lower fluorescence signal. As a consequence of the Poisson photon statistics, therefore the SNR in the recorded image will also be much lower than in normal confocal imaging. This means that also the normally straightforward deconvolution procedure for converting the PSF-scan pattern data into a proper image is more involved. Modern commercial and open-source (see, e.g., Waithe et al. 2016) software incorporates efficient algorithms, which apply, e.g., time-correlated photon counting and theoretical PSF-construction methods to reduce noise and thus increase the image contrast for better spatial resolution.
Comparison of STED, confocal and optical microscopy of ultra-short pitch cholesterics
Published in Liquid Crystals, 2020
J. Pišljar, G. Posnjak, S. Pajk, A. Godec, R. Podlipec, B. Kokot, I. Muševič
Topological defects and textures of liquid crystals, liquid crystal dispersions and liquid crystal colloids have been a subject of intense studies in the last two decades [1]. This interest was mainly driven by the extraordinary richness of fundamental topological phenomena observed in nematic and chiral nematic liquid crystals with or without inclusions. Defects and textures of liquid crystals are usually studied using polarised optical microscopy, which is limited to 2D images and finite optical resolution. The application of new microscopy methods, such as the Fluorescent Confocal Polarisation Microscopy (FCPM) [2], opened new and fascinating insight into the 3D director structures that could be visualised and reconstructed in amazing detail. Yet there is another class of super-resolution microscopies, which have not yet been studied in great detail in LCs. This includes Stimulated Emission Depletion (STED) microscopy, which is a variant of fluorescent microscopy. There are only a few studies of STED microscopy in LCs [3,4] and a single study of STED effect and its efficiency in the nematic and smectic-A liquid crystal [5]. It is therefore in the interest of liquid crystal community to compare the advantages, drawbacks and the practically achievable resolution of STED microscopy with standard optical and fluorescent microscopy when imaging structures and defects in liquid crystals.
Correction of dichroic mirror-induced PSF distortion in STED microscopy
Published in Journal of Modern Optics, 2018
Yanghui Li, Hui Zhou, Xiaoyu Liu, Chengliang Xia, Le Wang
Stimulated emission depletion (STED) microscopy, as one of several most popular types of super-resolution imaging techniques, has currently been well-known for its significant resolution improvements over confocal microscopy, since it was initially conceived by Stefan W. Hell in 1994 [1–3]. To break the diffraction limit, STED microscopy restricts the excited fluorescence everywhere except at the focal spot by stimulated emission. An ordinary excitation focal spot, nested by a ring-shaped depletion one, scans across the sample and the fluorescence is inversely collected by the same objective lens [2–5]. Before focused onto the photon detector, the fluorescence is separated from the common path by a dichroic mirror (DM) [3,6,7]. The optical properties of the DM are, therefore, very critical for the performance of the STED microscopy: its transmission and reflection spectra directly determine the power efficiency of the system. To optimize spectral curve of the DM, the DM is always designed and fabricated as the coating with multi-layer structure, and polarization difference between s- and p-polarized components is inevitable at large incident angle, which makes polarization maintenance difficult [8–10]. Related researches are still few, as the polarization distortion induced by the DM is negligible in most confocal microscopes. However, in STED microscopes, the imperfect polarization state may significantly reshape the depletion PSF, resulting in a distorted effective PSF and further a worse image quality [11–14].
Depletion of carbon dots in stimulated emission depletion microscopy developed with 405/532 nm continuous-wave lasers
Published in Journal of Modern Optics, 2022
Wenxuan Zhao, Shenghua Ma, Yueqiang Zhu, Chen Zhang, Xiaoqiang Feng, Wei Zhao, Guiren Wang, Kaige Wang
Optical microscopy is an essential detection technique in the imaging of microstructures, such as biological cells and tissues in life science. Owing to Abbe’s diffraction limit, conventional optical microscopy is not capable to distinguish the featured structures with sizes below , where is wavelength of light and is numerical aperture of the objective lens [1]. To break the diffraction barriers, several super-resolution techniques are proposed and developed [2] in the last two decades, e.g. photoactivated localization microscopy [3], stochastic optical reconstruction microscopy [4], stimulated emission depletion (STED) [5], saturated structured illumination microscopy [6] and others [7–9]. Among several techniques, STED has many unique advantages. By reducing the volume of effective fluorescence emission, STED microscopy shrinks the point spread function and breaks the diffraction barriers to achieve a spatial resolution below 30 nm [10] through a purely physical method without post-reconstruction and chemical reactions [9]. The lateral spatial resolution () of STED microscopy is determined by the following equation [11,12]: where denotes the maximum intensity of the STED beam and is the saturation intensity where the probability of fluorescence emission is reduced to a half of maximum. Therefore, increasing the ratio of the could effectively improve the spatial resolution. Developing highly efficient fluorescence probes with the low and excellent photobleaching resistance is essential for STED applications [13].