Three-dimensional super-resolution high-throughput imaging by structured illumination STED microscopy

Stimulated emission depletion (STED) microscopy is able to image fluorescence labeled samples with nanometer scale resolution. STED microscopy is typically a point-scanning method, limited by the high intensity requirement of the depletion beam. With the development of high peak power lasers, two dimensional parallel STED microscopy has been developed. Here, we develop the theoretical basis for extending STED microscopy to three dimensional imaging in parallel. This method uses structured illumination (SI) to generates a three dimensional depletion pattern. Compared to the two dimensional parallel STED microscopy, the 3D SI-STED microscopy generates intensity modulation along the light propagation direction without requiring higher laser power. This method not only achieves axial super-resolution of STED microscopy but also greatly reduces photobleaching and photodamage for 3D volumetric imaging.National Institutes of Health (U.S.) (NIH 1-U01-NS090438-01)National Institutes of Health (U.S.) (NIH 5-P41-EB015871)Hamamatsu Corporatio

Far-Field Optical Microscopy Based on Stimulated Emission Depletion

Conventional lens-based (far-field) fluorescence microscopy is a widely used imaging technique with spatial resolution up to 150–350 nm. However, this technology cannot discern very small structural features, because the spatial resolution is limited by diffraction to about half of the wavelength of light (λ/2，λ is the wavelength of light). Hence, most of the developments in microscopy aim at improving resolution. In the past decades, stimulated emission depletion (STED) microscopy has been developed to bypass the diffraction limit for the application in biological imaging with resolution approaching the nanoscale. The basic principle of STED microscopy is to employ a doughnut-shape laser called the depletion laser which inhibits fluorescence emission and improves the resolution of the focal plane by depleting the peripheral fluorescence. Thereby, STED microscopy avoids the diffraction barrier and improves the spatial resolution. STED microscopy has been widely applied to address many problems in biology with both continuous wave and pulsed wave lasers. Various fluorescent nanoparticles, therefore, are attractive for far-field super-resolution microscopy. During the past decades, fluorescent nanoparticles have been used as a fluorescent label, fluorescent probe or marker for super-resolution imaging in vitro andvivo. In our study, STED microscopy is one of the breakthrough technologies that belongs to far-field optical microscopy and can reach the nanoscale spatial resolution. We demonstrate a far-field optical microscopy based on pulsed-wave lasers with the violet (405 nm) and green lasers (532 nm) for excitation and STED, respectively. Firstly,fluorescent dye - Coumarin 102 is applied to verify the stability and reliability of the STED microscopy. Then, one suitable nanoparticle is selected from three different kinds of nanoparticles (Silica Nanoparticles-NFv465, flouro-Max blue aqueous fluorescent nanoparticles, light yellow nanoparticles) based on their absorption and depletion spectrum and depletion efficiency under different depletion power. Light yellow fluorescent nanoparticles (LYs) are selected for characterizing the spatial resolution of the STED microscopy. Finally, the laser beams of the STED microscopy are utilized to scan along a glass slide, which is coated with the LYs. A two-dimensional image of the LYs pattern is established and compared with the confocal imaging, indicating that a spatial resolution (approximately 76.02 nm) has been obtained in the STED imaging so far. Even though the resolution of STED microscopy with pulsed-laser has the room to be improved, the present work shows that our lab has successfully built up the STED microscopy with the pulsed-laser

New Concepts for STED Microscopy

Fluorescence nanoscopy allows to non-invasively resolve three-dimensional cellular structures beyond the diffraction limit. One of these high resolution imaging techniques is stimulated emission depletion (STED) microscopy. However, the practically achievable resolution of a STED microscope is often limited by photobleaching. One method to overcome this limitation is tomographic STED (tomoSTED) microscopy. In tomoSTED microscopy, excited fluorophores in the sample plane are depleted by 1D STED patterns, which lead to an effective narrowing of the fluorescence-allowed area in a single direction. As the effective 1D STED-PSFs exhibit both a higher resolution in the respective direction as well as a higher signal as compared to conventional STED, the STED laser power as well as the exposure time can be reduced, leading to a lower light dose. A highly resolved image in two dimensions is reconstructed from multiple images, each exhibiting a different orientation of the 1D STED-PSF. The number of required pattern orientations depends in this context on the ratio of the resolutions in the depleted and non-depleted direction. Since the resolution per pattern orientation is only increased along a single direction, imaging along the other direction is still diffraction-limited. Therefore, the resolution along this direction can be increased by utilizing the concept of image scanning microscopy (ISM) and the number of required pattern orientations can be accordingly reduced. This leads to a lower overall acquisition time and translates directly into a lower light dose. Within this thesis, the combination of tomoSTED and ISM was investigated for the first time, both in theory and experiment. Furthermore, it was investigated whether the tomoSTED principle can be extended from 2D to 3D.2022-01-1

Super-Resolution STED Microscopy in live Brain Tissue

STED microscopy is one of several fluorescence microscopy techniques that permit imaging at higher spatial resolution than what the diffraction-limit of light dictates. STED imaging is unique among these super-resolution modalities in being a beam-scanning microscopy technique based on confocal or 2-photon imaging, which provides the advantage of superior optical sectioning in thick samples. Compared to the other super-resolution techniques that are based on widefield microscopy, this makes STED particularly suited for imaging inside live brain tissue, such as in slices or in vivo. Notably, the 50nm resolution provided by STED microscopy enables analysis of neural morphologies that conventional confocal and 2-photon microscopy approaches cannot resolve, including all-important synaptic structures. Over the course of the last 20years, STED microscopy has undergone extensive developments towards ever more versatile use, and has facilitated remarkable neurophysiological discoveries. The technique is still not widely adopted for live tissue imaging, even though one of its particular strengths is exactly in resolving the nanoscale dynamics of synaptic structures in brain tissue, as well as in addressing the complex morphologies of glial cells, and revealing the intricate structure of the brain extracellular space. Not least, live tissue STED microscopy has so far hardly been applied in settings of pathophysiology, though also here it shows great promise for providing new insights. This review outlines the technical advantages of STED microscopy for imaging in live brain tissue, and highlights key neurobiological findings brought about by the technique.The authors acknowledge funding for their general work from the Spanish Ministry of Science and Innovation (SAF-2017-83776-R, RYC-2014-15994 and IJCI-2017-32114), the Basque Government (PIBA19-0065 and PIBA-2020-1-0061), and the University of the Basque Country (GIU18/094 and INF19-29