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We demonstrate nanoscale resolution in far-field optical microscopy based on photo-switching of molecules. By enabling, recording and disabling fluorescence from individual labels sequentially, the detection volume is reduced to the size of a single molecule and the diffraction limit is broken. Images of nanostructures milled into a coverslip and tagged by fluorescent proteins could be recorded at 50 nm resolution. Due to the fast and asynchronous image acquisition protocol used in these experiments, we were able to reduce acquisition times to ∼2.5 min, which is two orders of magnitude lower than in previous implementations. [ABSTRACT FROM AUTHOR]

We demonstrate two-color far-field fluorescence microscopy with nanoscale spatial resolution based on the photoswitching of individual fluorescent markers. By enabling, recording, and disabling the emission of the reversibly switchable fluorescent protein rsFastLime and of the organic fluorophore cyanine5, we recorded two-color nanoscale images inside whole cells. The position of individual emitters was determined with a typical accuracy of 20 nm, which largely constitutes the lateral resolution of the system. Photoswitching in two-color colocalization experiments represents a major step towards the application of far-field fluorescence nanoscopy to the study of (biological) samples on the macromolecular level. [ABSTRACT FROM AUTHOR]

Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S[sub 1] with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100 ps were generated by convolution with the excitation pulse. The lifetime of the S[sub 1] state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79±0.33 ns (RB) and 3.79±0.38 ns (R6G) in aqueous solution. [ABSTRACT FROM AUTHOR]