PcSOFI as a smart label-based superresolution microscopy technique.
Stochastic optical fluctuation imaging (SOFI) is a superresolution imaging technique that uses the flickering of fluorescent labels to generate a microscopic image with a resolution better than what the diffraction limit allows. Its adaptation towards fluorescent protein-labeled samples (called photoconversion SOFI or pcSOFI) allows for a straightforward and easily accessible way of generating superresolution images. In this protocol, we will discuss how so-called "smart labels," and specifically the reversibly switchable fluorescent proteins, have opened doors towards superresolution imaging in general and we provide a protocol on how to perform pcSOFI on HeLa cells expressing human β-actin labeled with the reversibly photoswitchable fluorescent protein Dronpa.
Department of Chemistry, University of Leuven, Celestijnenlaan 200 F, 3001, Heverlee, Leuven, Belgium.
Benjamien
B
Moeyaert
Peter
P
Dedecker
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D057230:Limit of Detection; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D012984:Software
D000199:Actins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins