PNNL

Abstract

Fluorescence imaging has transformed biological sciences and opened a window to reveal biological mechanisms in real time despite Abbe’s diffraction limit restricts current microscope resolution to 300 nm?.HDH2 Recently, two high-resolution fluorescence microscopic techniques emerged: one uses a special photoactivatable green fluorescent proteinHDH3 and the other employs a pair of cy3/cy5 dyes.HDH4 Both avoid Abbe’s diffraction limit by photoswitching nearby fluorophores off. Thus, photoswitching fluorescence between a bright and a dark state promises to deliver a wealth of information regarding biological phenomena at the nanoscale. The ideal probe is a key-enabling single molecule that can be photoswitched on and off. Such wonderful properties, albeit implausible to imagine at first, were realized in spiropyran derivatives. While being photoswitched, one molecule alternates red-fluorescence on-and-off. Using such photo-actuated unimolecular logical switching attained reconstruction (PULSAR) microscopy, we achieved high-resolution fluorescence imaging down to 80 nm? in nanostructures and cellular organelles.