Synchronously amplified fluorescence image recovery (SAFIRe).

Fluorescence intermittency severely limits brightness in both single molecule and bulk fluorescence. Herein, we demonstrate that optical depopulation of organic fluorophore triplet states opens a path to significantly increased sensitivity by simultaneously increasing brightness and greatly reducing background through synchronously detected fluorescence modulation. Image recovery is achieved through selective fluorescence enhancement via modulating a secondary laser excitation at much lower energy than the observed emission in order to depopulate the long-lived triplet states. A series of xanthene dyes that exhibit efficient triplet-state formation demonstrate that this method of selective signal extraction can be achieved at moderate primary and secondary excitation intensities through tailoring dye photophysics and imaging conditions. Up to 5-fold increases in solution-based fluorescence over primary laser excitation alone was achieved upon secondary laser excitation, and dynamic control of signal modulation was demonstrated over a wide time range simply by varying the modulation frequency of the laser used for depopulation of the triplet state. We identify the photophysical characteristics that enable existing or to-be-designed fluorophores to be used in synchronously amplified fluorescence image recovery (SAFIRe) microscopy.