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

Autor:	Masch JM; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.; Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37073 Göttingen, Germany., Steffens H; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany., Fischer J; Optical Nanoscopy Division, German Cancer Research Center, 69120 Heidelberg, Germany., Engelhardt J; Optical Nanoscopy Division, German Cancer Research Center, 69120 Heidelberg, Germany.; Department of Optical Nanoscopy, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany., Hubrich J; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany., Keller-Findeisen J; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany., D'Este E; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany., Urban NT; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.; Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37073 Göttingen, Germany., Grant SGN; Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh EH16 4SB, United Kingdom., Sahl SJ; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany., Kamin D; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; dirk.kamin@mpibpc.mpg.de stefan.hell@mpibpc.mpg.de.; Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37073 Göttingen, Germany., Hell SW; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; dirk.kamin@mpibpc.mpg.de stefan.hell@mpibpc.mpg.de.; Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37073 Göttingen, Germany.; Optical Nanoscopy Division, German Cancer Research Center, 69120 Heidelberg, Germany.; Department of Optical Nanoscopy, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2018 Aug 21; Vol. 115 (34), pp. E8047-E8056. Date of Electronic Publication: 2018 Aug 06.
DOI:	10.1073/pnas.1807104115
Abstrakt:	Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes. Competing Interests: The authors declare no conflict of interest. (Copyright © 2018 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
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