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.
Highlights
Neuronal synapses are the most basic functional units of the brain
We evaluated 14 different red and far-red fluorescent proteins (FP) fused to Lifeact in CV-1 cells (African green monkey Cercopithecus aethiops kidney cells), in terms of their suitability for STED microscopy (Supplementary Table S1)
No signs of phototoxicity were observed, at either 560 or 586 nm excitation, nor with the 732 or 775 nm STED light, which renders these ranges of wavelengths ideal for in vivo STED microscopy
Summary
Neuronal synapses are the most basic functional units of the brain. The postsynaptic part is often placed on little dendritic protrusions, the dendritic spines. Spine dynamics are best observed in the living brain, where the neuronal network remains intact and native synaptic structures are preserved Because of their minute size of 0–2 μm in length, far-field light microscopy is the only technique to visualize spines in the living tissue or organism. We set out to study the usability of red emitting fluorescent proteins (FP) for in vivo STED microscopy through actin labelling, which is ubiquitously expressed in dendrites and spines. MNeptune[2] proved to be a good alternative for live cell STED microscopy with far-red emitting proteins, we experienced difficulties in labelling of the living mouse; proximal dendrites were much brighter than distal dendrites, rendering fluorescence imaging of layer 1 dendrites challenging. We found that both actin labels, Lifeact and Actin-Chromobody, have an influence on actin morphology at high expression levels and must be deployed cautiously
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