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Published July 25, 2016 | Published
Journal Article Open

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast


Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells.

Additional Information

© 2014 SPIE. Paper 140362R received Jun. 9, 2014; revised manuscript received Jul. 31, 2014; accepted for publication Jul. 31, 2014; published online Aug. 21, 2014. The authors appreciate the close reading of the manuscript by Professor James Ballard. We thank Lidai Wang, Konstantin Maslov, and Liren Zhu for helpful discussions. This work was supported in part by the Grant Nos. EB016986 (NIH Director's Pioneer Award), R01 CA186567 (NIH Director's Transformative Research Award), S10 RR028864, and CA159959 (to L.V.W.), and GM073913 and CA164468 (to V.V.V.) from the National Institutes of Health.

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