TY - JOUR
T1 - Correlated confocal and super-resolution imaging by VividSTORM
AU - Barna, László
AU - Dudok, Barna
AU - Miczán, Vivien
AU - Horváth, András
AU - László, Zsófia I.
AU - Katona, István
N1 - Funding Information:
acknoWleDGMents The authors are grateful to B. Pintér for technical assistance and help with establishing the tissue handling protocol; E. Tischler for technical assistance; and E. Szabadits and R. Nyilas (Institute of Experimental Medicine, Hungarian Academy of Sciences) for help with optimizing the immunostaining. We also thank M. Ledri (Institute of Experimental Medicine, Hungarian Academy of Sciences) for providing brain samples, I. Kacskovics (Immunogenes, Ltd.) and G. Balla (Institute of Experimental Medicine, Hungarian Academy of Sciences) for providing antibodies, and A. Zimmer (Institute of Molecular Psychiatry, University of Bonn) for providing knockout mice. We are grateful to S. Turbucz for his help with coding the software. We acknowledge the help of the Nikon Microscopy Center at the Institute of Experimental Medicine, Nikon Europe, Nikon Austria and Auro-Science Consulting for kindly providing microscopy support. The authors are indebted to I. Sase and I. Burda from Nikon for their invaluable help with the application of N-STORM. This work was supported by the Hungarian Academy of Sciences Momentum Program LP-54/2013, by the European Research Council Starting grant no. 243153 and by the Wellcome Trust International Senior Research Fellowship (to I.K.).
Publisher Copyright:
© 2015 Nature America, Inc. All rights reserved.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2015/12/30
Y1 - 2015/12/30
N2 - Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the life sciences as an efficient approach to visualize molecular distribution with nanoscale precision. However, it has been challenging to obtain and analyze such data within a cellular context in tissue preparations. Here we describe a 5-d tissue processing and immunostaining procedure that is optimized for SMLM, and we provide example applications to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated confocal and 3D-superresolution imaging on these sections, which allows the visualization of nanoscale protein localization within labeled subcellular compartments of identified target cells in a few minutes. Finally, we describe the use of VividSTORM (http://katonalab.hu/index.php/vividstorm), an open-source software for correlated confocal and SMLM image analysis, which facilitates the measurement of molecular abundance, clustering, internalization, surface density and intermolecular distances in a cell-specific and subcellular compartment-restricted manner. The protocol requires only basic skills in tissue staining and microscopy.
AB - Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the life sciences as an efficient approach to visualize molecular distribution with nanoscale precision. However, it has been challenging to obtain and analyze such data within a cellular context in tissue preparations. Here we describe a 5-d tissue processing and immunostaining procedure that is optimized for SMLM, and we provide example applications to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated confocal and 3D-superresolution imaging on these sections, which allows the visualization of nanoscale protein localization within labeled subcellular compartments of identified target cells in a few minutes. Finally, we describe the use of VividSTORM (http://katonalab.hu/index.php/vividstorm), an open-source software for correlated confocal and SMLM image analysis, which facilitates the measurement of molecular abundance, clustering, internalization, surface density and intermolecular distances in a cell-specific and subcellular compartment-restricted manner. The protocol requires only basic skills in tissue staining and microscopy.
UR - http://www.scopus.com/inward/record.url?scp=84988566258&partnerID=8YFLogxK
U2 - 10.1038/nprot.2016.002
DO - 10.1038/nprot.2016.002
M3 - Article
C2 - 26716705
AN - SCOPUS:84988566258
SN - 1754-2189
VL - 11
SP - 163
EP - 183
JO - Nature Protocols
JF - Nature Protocols
IS - 1
ER -