Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging

Abstract

Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.

Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence

“…Photobleaching is not only inconvenient, but can limit observations, while phototoxicity can lead to artifactual observations (Boudreau et al, 2016; Han et al, 2017; Icha et al, 2017; Tinevez et al, 2012; Waldchen et al, 2015). Over the years, improved fluorochromes, camera and photodetector sensitivity, computer algorithms, and elegant design of dyes with suitable physicochemical properties have reduced the light intensity and/or exposure needed to obtain good signal-to-noise (Boutros et al, 2015; Jing et al, 2021; Lidke and Lidke, 2012). Despite these efforts, photobleaching and phototoxicity remain significant problems in live-cell imaging.…”

“…Live-cell fluorescence microscopy is an essential tool to determine and characterize the spatio-temporal dynamics of molecules, supra-molecular assemblies, organelles, and of entire cells (Jensen, 2013; Lidke and Lidke, 2012). Over the years, technological enhancements have emerged that pushed the boundary in the spatial resolution of cellular organization, the detection limits of cameras and photo-detectors allowing for single-molecule imaging, and image analysis that permits high-throughput, high-content and unbiased quantification (Boutros et al, 2015; Jing et al, 2021; Lidke and Lidke, 2012; Shashkova and Leake, 2017). These improvements depended and continue to depend on innovative manipulation of the physico-chemical properties of fluorochromes, hardware advances leading to superior camera sensitivity and detection, and a surge in computational power and better algorithms (Boutros et al, 2015; Lidke and Lidke, 2012; Moen et al, 2019; Ouyang et al, 2018; Peterson et al, 2016; Rust et al, 2006; Shtengel et al, 2009).…”

Cover Feature: Smart Photochromic Materials Triggered with Visible Light (Eur. J. Org. Chem. 19/2022)

“…[187] Structural optimization of photoswitchable fluorophores [188] towards compatibil-ity with aqueous environment [189] and activation with visible light (e. g. in spirooxazines [190] or diarylethenes [191] ) could expand the scope of this technology in complex biological setups. [192] Optogenetics is a technique to control activity of neurons or other cell types with light, by expression of light-sensitive ion channels, pumps, or enzymes. [193] The analogous effect -photocontrol of ion channel activity -can be achieved with molecular photoswitches.…”

Imaging Endocytosis Dynamics in Health and Disease

“…In Vivo Imaging and Its Applications to Visualize Membrane Trafficking Despite significant progress in SRM techniques, their application in living samples are limited due to poor imaging depth. Considerable efforts have been extended to enhancing the imaging depth and developing highly-sensitive fluorescent probes to realize real-time imaging in vivo [126]. For example, Adaptive Optics (AO) has been introduced in many SRM techniques to enhance the imaging depth by eliminating sample-induced distortions [127].…”