Recently, super-resolution techniques have been employed to study their mechanistic and functional characteristics at a single-molecule level.Yingying Jing, Chenshuang Zhang, Bin Yu, Danying Lin* and Junle Qu*Super-resolution techniques aim to increase the resolution of images by adding detail. Viral particles vary greatly in size, but are typically below the resolution limit of conventional light microscopy. EDT, Sunday, June 13, 2021) A new imaging technique has the potential to detect neurological disorderssuch as Alzheimer’s diseaseat their earliest stages, enabling physicians to diagnose and. New super-resolution technique allows for more detailed brain imaging Published by Ajisebutu Doyinsola Reston, VA (Embargoed until 5:00 p.m.These SRM techniques are most prominently harnessed in two distinct families: one employs patterned illumination to spatially modulate the fluorescence behavior and their related derivatives, such as stimulated emission depletion microscopy (STED) ( Hell and Wichmann, 1994 Vicidomini et al., 2018), structured illumination microscopy (SIM) ( Gustafsson, 2000 Li et al., 2015 Turcotte et al., 2019) and reversible saturable optically linear fluorescence transitions (RESOLFTs) ( Grotjohann et al., 2011) the other obtains super-resolution images based on the localization of individual emitting molecules, such as stochastic optical reconstruction microscopy (STORM) ( Rust et al., 2006 Bates et al., 2013) and photoactivated localization microscopy (PALM) ( Betzig et al., 2006 Henriques et al., 2011). Hence, sub-diffraction imaging techniques are needed urgently for investigating the fine structures in vivo.Over the past 2 decades, advances in super-resolution microscopy (SRM) have revolutionized the field of fluorescent imaging and become valuable tools in biological studies ( Coltharp and Xiao, 2012 Baddeley and Bewersdorf, 2018 Schermelleh et al., 2019). Nevertheless, more detailed measurement of morphological changes in living species has been hindered by the diffraction limitation of light, which is about half the wavelength of light (200–300 nm) ( Hell et al., 2004 Agrawal et al., 2013).Besides, the imaging rates are still insufficient for many in vivo applications, especially those involving signal transportation studies ( Galbraith and Galbraith, 2011 Biteen and Willets, 2017). The unnecessary excitation of out-of-focus fluorophores degrades the quality of the in-focus signal, making it difficult to resolve the fine structures. Et al., 2020).Despite significant progress, the application of present SRM techniques to scattering tissues or living samples are limited due to poor imaging depth.As an extended technique based on confocal microscopy, STED could obtain the images directly, and thus is better suited for in vivo imaging ( Jahr et al., 2020 Steffens et al., 2020). Scanning the sharpened PSFs across the sample could allow recording a super-resolved image with high resolution. On that account, the depletion beam needs an illumination pattern shaped like doughnut with non-zero intensity at the periphery and zero intensity at the center of the excitation spot. Briefly, STED microscopy is a two-beam technique that applies a STED beam to suppress the emission of the fluorescent molecules located off the core of the excitation region in order to sharpen the effective point spread function (PSF) ( Figure 1A) ( Yang et al., 2016). Super-Resolution Techniques for In Vivo Imaging Stimulated Emission Depletion (STED) MicroscopyIn 1994, the concept of STED microscopy was first proposed and it was subsequently demonstrated experimentally ( Hell and Wichmann, 1994). Then, the applications of these techniques in various biological areas are discussed, and the challenges as well as future trends in this emerging field are highlighted.
(B) Schematic drawing of the upright scanning STED for in vivo imaging ( Berning et al., 2012). (2016) with permission from Royal Society of Chemistry. Reprinted from Yang et al. The combination of excitation laser and donut-shaped STED laser could decrease the size of PSF effectively ( Yang et al., 2016). (A) The principle of STED technique. Overview of modified STED microscopy for in vivo imaging. Campground layout ideas(2018) with permission from eLife Sciences. Reprinted from Pfeiffer et al. The 2P image depicted dendrites of pyramidal neuron in hippocampal CA1 region. (C) Diagram of two photon (2P) STED microscope ( Pfeiffer et al., 2018). The STED image of dendritic and axonal structures was obtained from a living mouse brain under the objective lens (1.3 NA, glycerol immersion). (2012) with permission from American Association for the Advancement of Science. (2021) with permission from Optical Society of America.Because of specimen-induced scattering distortion and aberrations, there is a great challenge for STED microscopy to maintain consistent resolution in deep tissue inside living bodies. Reprinted from Velasco et al. Super-resolution image showed a 3D dendrite at 76 µm below the cortical surface. The microscopy equipped with a double-pass spatial light modulator (SLM) to impart both the vortex and top-hat phase masks on the depletion beam. They developed an upright scanning STED microscope with a 1.3 NA objective lens focusing 488 nm wavelength in a mouse brain ( Figure 1B). Afterwards, the real in vivo STED microscopy was first demonstrated also by his group in 2012 ( Berning et al., 2012). They could image the actin at a depth of 120 μm below the tissue surface inside living brain slices with a spatial resolution of 60–80 nm ( Urban et al., 2011). Nvidia geforce 1650 drivers downloadFor example, Kebin Shi’s group reported a Gaussian-Bessel STED (GB-STED) microscopy, which achieved an imaging depth of 100 μm in brain tissue by modulating the depletion beam into a hollow Bessel beam and using a conventional Gaussian beam for excitation ( Yu et al. Later, a few groups also modified STED technique to increase the imaging depth through various methods. This STED technique was successfully applied to image the 10–15 μm molecular layer below the surface with a resolution of about 70 nm.
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