Diffraction-unlimited fluorescence microscopy of living biological samples using pcSOFI.
The complex microscopic nature of many live biological processes is often obscured by the diffraction limit of light, requiring diffraction-unlimited fluorescence microscopy to resolve them. Because of the vast range of different processes that can be studied, sub-diffraction imaging should work efficiently under many different conditions. Photochromic stochastic optical fluctuation imaging (pcSOFI) is a recent addition to the field of diffraction-unlimited fluorescence microscopy. This robust and versatile method employs a statistical analysis of random fluctuations in the emission of single labels, in this case reversibly switchable fluorescent proteins (RSFPs), to retrieve super-resolution information. Added to the resolution enhancement, pcSOFI also offers contrast enhancement and background reduction in a practical and convenient way. Here, we describe the necessary steps to obtain diffraction-unlimited images, including multicolor and three-dimensional imaging, and highlight the advantages of pcSOFI together with the circumstances under which pcSOFI can be favorably applied.
Department of Chemistry, University of Leuven, Heverlee, Belgium.
Sam
S
Duwé
Benjamien
B
Moeyaert
Peter
P
Dedecker
D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
D005456:Fluorescent Dyes; D011506:Proteins
3D imaging; fluorescence imaging; pcSOFI; reversible photoswitchable fluorescent proteins; super-resolution microscopy