Molecules and methods for super-resolution imaging.
By looking at a fluorescently labeled structure one molecule at a time, it is possible to side-step the optical diffraction limit and obtain "super-resolution" images of small nanostructures. In the Moerner Lab, we seek to develop both molecules and methods to extend super-resolution fluorescence imaging. Methodologies and protocols for designing and characterizing fluorophores with switchable fluorescence required for super-resolution imaging are reported. These fluorophores include azido-DCDHF molecules, covalently linked Cy3-Cy5 dimers, and also the first example of a photoswitchable fluorescent protein, enhanced yellow fluorescent protein (EYFP). The imaging of protein superstructures in living Caulobacter crescentus bacteria is used as an example of the power of super-resolution imaging by single-molecule photoswitching to extract information beyond the diffraction limit. Finally, a new method is described for obtaining three-dimensional super-resolution information using a double-helix point-spread function.
Thompson
Michael A
MA
Department of Chemistry, Stanford University, Stanford, California, USA.
Biteen
Julie S
JS
Lord
Samuel J
SJ
Conley
Nicholas R
NR
Moerner
W E
WE
D000818:Animals; D001386:Azides / Q000737:chemistry; D001419:Bacteria / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D010777:Photochemistry; D017785:Photons
D001386:Azides; D005456:Fluorescent Dyes