In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins

Abstract

The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

In vivo STED microscopy visualizes PSD95 sub-structures and morphological changes over several hours in the mouse visual cortex

“…Therefore we assume that the large assemblies belong to mushroom spines, which indeed needs to be proven in the future by an additional labelling of the spine morphology in the living mouse. This could be verified by using either a cytosolic spine volume-, or actin labelling 21 . However, we can assume that the spine head is at least in the size of the PSD95 assembly.…”

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

“…The NIR FPs are excited at or close to their peak by 640-670 nm lasers. In contrast, the far-red GFP-like FPs previously used for live-cell STED, such as E2-Crimson 34 , mGarnet 35 , TagRFP657 36 and mNeptune2 37,38 , have to be excited at the red edge of their spectrum. Moreover, the far-red FPs suffer from lower photostability, allowing to take no more than 20 consecutive frames without loss in image quality 35,39 , as compared to 50 frames obtained with emiRFPs.…”

Super-resolution microscopy as a powerful tool to study complex synthetic materials

“…Altogether SRM has led to remarkable improvement in the assessment of the intracellular localization and trafficking of nanoparticles compared to conventional fluorescence microscopy, being a valuable tool to dissect internalization pathways in cultured cells and, potentially, in vivo. 130,131 [H2] Material stability The enhanced resolution of SRM allows us to go beyond the bare visualization of nano-objects and their location, and probe several material properties in the cellular environment. This is particularly relevant for complex molecular architectures, designed to be responsive and adaptive in the biological environment.…”