Nanoscopy in a Living Mouse Brain

Abstract

We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

“…Scanning of such a reduced observation volume produces a fluorescence image with superior spatial resolution. STED microscopy is well suited for (dynamic) imaging in live cells 26 and even in vivo 27 . Apart from a fluorescent protein with a limited fluorescence quantum yield and photostability 28 , no appropriate near-infrared fluorophores for STED microscopy of intracellular proteins in living cells are available currently.…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…The STED approach can be extended to a more general principle as shown in reversible saturable/switchable optically linear fluorescence transition (RESOLFT) microscopy [76], which can be performed with multiple kinds of switchable fluorescent molecules, slightly reducing the intensity needed for imaging. With STED a resolution of 70 nm has been reached in living cells [65,77], as can be seen in Figure 3E where a two-color STED microscope is used to image a living HEK293 cell over an interval of more than 4 minutes [65]. Drawbacks of targeted switching techniques such as STED and RESOLFT are that they often require high intensities, resulting in high photo-bleaching and photo-toxic effects.…”

Fluorescence nanoscopy. Methods and applications

“…Video-rate (28 Hz) STED imaging at 65 nm lateral resolution has been demonstrated on living cells, although the field of view was relatively small (4.5 μm 2 ), and it was confined to two dimensions possibly due to out-of-focus photodamage, which precluded from whole-cell imaging [26,27]. In an impressive demonstration, in vivo STED of the brain of a living mouse has been recently demonstrated [28]. STED live-cell imaging can be performed using synthetic fluorophores, which are advantageous as they are brighter and more photostable than fluorescent proteins.…”

“…Fluorescence nanoscopy makes possible the investigation of the nanoscale dynamic organisation of proteins within subsynaptic domains: The fate of synaptic vesicles after exocytosis and fusion with the membrane has been studied by STED microscopy in fixed [199,200] and live neurons using video-rate (20)(21)(22)(23)(24)(25)(26)(27)(28) [27,201] and time-lapse STED microscopy [202,203]. AMPA receptors trafficking in neuronal cells have been investigated by single particle tracking PALM [204].…”

“…Going one step farther, STED has been demonstrated on the living brain of an anaesthetised mouse expressing YFP as a nonfusion protein in the neuronal cytoplasm [28]. The high average power required for STED imaging hinders, however, its application to live tissue.…”