title: Correlated confocal and super-resolution imaging by VividSTORM.
abstract: 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.
authors.affiliation: Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Barna
authors.affiliation: Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.|School of Ph.D. Studies, Semmelweis University, Budapest, Hungary.
authors.forename: Barna
authors.identifier: http://orcid.org/0000-0001-5078-0090
authors.initials: B
authors.lastname: Dudok
authors.affiliation: Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.|Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary.
authors.forename: Vivien
authors.initials: V
authors.lastname: Miczán
authors.affiliation: Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Horváth
authors.affiliation: Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.|School of Ph.D. Studies, Semmelweis University, Budapest, Hungary.
authors.forename: Zsófia I
authors.initials: ZI
authors.lastname: László
authors.affiliation: Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: István
authors.identifier: http://orcid.org/0000-0003-2808-3330
authors.initials: I
authors.lastname: Katona
mesh_terms: D000465:Algorithms; D000818:Animals; D001692:Biological Transport; D016000:Cluster Analysis; D021621:Imaging, Three-Dimensional; D051379:Mice; D008853:Microscopy / Q000379:methods*; D018613:Microscopy, Confocal; D057054:Molecular Imaging / Q000379:methods*; D059629:Signal-To-Noise Ratio; D012984:Software; D013194:Staining and Labeling; D014584:User-Computer Interface