Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling

Abstract

SignificanceIn vivo fluorescence microscopy with resolution well beyond the diffraction limit entails complexities that challenge the attainment of sufficient image brightness and contrast. These challenges have so far hampered investigations of the nanoscale distributions of synaptic proteins in the living mouse. Here, we describe a combination of stimulated emission depletion microscopy and endogenous protein labeling, providing high-quality in vivo data of the key scaffolding protein PSD95 at the postsynaptic membrane, which frequently appeared in extended distributions rather than as isolated nanoclusters. Operating in the far-red to near-IR wavelength range, this combination promises reduced photostress compared with prior in vivo nanoscopy at much shorter wavelengths.

3D super-resolution deep-tissue imaging in living mice

“…For our in vivo imaging experiments, we combined the use of organic dyes, Halo-tags, and rAAV technology to label neurons deep in the living mouse brain. While live-cell compatible red organic dyes like ATTO590 offer superior STED resolution and photostability over fluorescent proteins of the same color [36], the use of rAAVs offers flexibility in the labelling scheme. The virus can be easily modified to target a different cell type, to label specific proteins (e.g.…”

“…by expressing the Halo-tags fused to a protein of interest), and to additionally express SNAP-tags so that a second live-cell compatible dye (e.g. silicon rhodamine [36,40]) can be used for multicolor superresolution imaging. To simplify future experiments, transgenic mouse lines that express SNAPand/or Halo-tags constitutively [36] offer a user-friendly alternative.…”

“…silicon rhodamine [36,40]) can be used for multicolor superresolution imaging. To simplify future experiments, transgenic mouse lines that express SNAPand/or Halo-tags constitutively [36] offer a user-friendly alternative. Moreover, instead of topically applying the cell-permeable dye to the exposed brain surface, one may consider intravenously injecting dyes that are able to cross the blood-brain barrier [41].…”

“…For these experiments, we required an in vivo labelling procedure that targeted neurons in the brain with the far-red live-cell compatible dye ATTO590. It has previously been shown that labelling with organic dyes emitting in the red to far-red range is advantageous for in vivo STED microscopy, as these dyes exhibit superior photophysical properties compared to their red fluorescent protein counterparts [36]. In line with this finding, we used a wild-type CD1 mouse line and recombinant adeno-associated virus (rAAV, serotype 2) infection to induce the expression of fused cytosolic GFP and Halo-tags [37] in a subset of cortical neurons ( Fig.…”

SEQUIN multiscale imaging of mammalian central synapses reveals loss of synaptic microconnectivity resulting from diffuse traumatic brain injury

“…The exceptional resolution enabled by single molecule localization microscopy approaches (STORM 74 , PALM 75,76 , and related techniques) and stimulated emission depletion (STED) 77 microscopy can reveal nanoscale features of individual chemical synapses challenging or impossible to detect with image scanning microscopy (e.g., subsynaptic protein nanoclusters 72,78 ). STED was also recently shown to be amenable to imaging postsynaptic structures in vivo through the use of a knock-in HaloTag fusion to PSD-95 and intracortical injection of a STED-appropriate fluorescent ligand 79 . SEQUIN analysis of natively-fluorescent synaptic fusion proteins ( Fig.…”