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

被引：80
作者：
Wegner, Waja [1 ,2 ,3 ]
Ilgen, Peter [1 ,2 ,3 ]
Gregor, Carola [4 ]
van Dort, Joris [1 ,3 ]
Mott, Alexander C. [1 ,3 ]
Steffens, Heinz [1 ,2 ,3 ,4 ]
Willig, Katrin I. [1 ,2 ,3 ]
机构：
[1] Univ Med Ctr Gottingen, Ctr Nanoscale Microscopy & Mol Physiol Brain, Opt Nanoscopy Neurosci, Gottingen, Germany
[2] Univ Gottingen, Collaborat Res Ctr 889, Gottingen, Germany
[3] Max Planck Inst Expt Med, Gottingen, Germany
[4] Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany
来源：
SCIENTIFIC REPORTS | 2017年 / 7卷
关键词：
DENDRITIC SPINES; NERVOUS-SYSTEM; F-ACTIN; NANOSCOPY; EXPRESSION; DYNAMICS; LIFEACT; REORGANIZATION;
D O I：
10.1038/s41598-017-11827-4
中图分类号：
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号：
07 ; 0710 ; 09 ;
摘要：
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 similar to 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.
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页数：10
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