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.
Richards
Chris I
CI
School of Chemistry and Biochemistry and Petit Institute for Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA.
Hsiang
Jung-Cheng
JC
Dickson
Robert M
RM
D004801:Eosine Yellowish-(YS) / Q000737:chemistry; D004923:Erythrosine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012395:Rose Bengal / Q000737:chemistry
D005456:Fluorescent Dyes; D012395:Rose Bengal; D004923:Erythrosine; D004801:Eosine Yellowish-(YS)