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RESOLFT was introduced to break the limited resolution in fluorescence microscopy given by thephysical limit of diffraction. RESOLFT needs saturable fluorescence depletion and illumination with spatial regions of zero intensity. Improvements in RESOLFT are still essential since (i) photobleaching restricts the use of large intensities obligate for high resolution, (ii) long acquisition times emerge from single-point scanning, and (iii) RESOLFT puts ahead special requirements on the dye´s photophysical properties. To counteract these problems, this work presents a RESOLFT-type microscope implementing parallelized ground state depletion (GSD). Parallelization allows rather fast image acquisition applying structured illumination combined with widefield detection on a camera. The depletion of the fluophore´s ground state, and thus the fluorescence, is realized using a pump-probe illumination scheme. To increase photostability, the sample was evacuated and cooled to approximately 80 K. Since the population of the triplet state is an intrinsic property of almost every dye and is enhanced by evacuation and low temperatures, parallelized GSD is generally applicable to a wide range of fluorescent markers. Applying parallelized RESOLFT an enhancement in resolution was proven for a bead sample as well as inside a cell. Parallelized GSD microscopy further prevents photostress on both fluorescent label and sample, since it encounters moderate illumination intensities < kW/cm2.
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530 Physik
530 Physics