title: Optical modulation and selective recovery of Cy5 fluorescence.
abstract: Fluorescence modulation offers the opportunity to detect low-concentration fluorophore signals within high background. Applicable from the single-molecule to bulk levels, we demonstrate long-wavelength optical depopulation of dark states that otherwise limit Cy5 fluorescence intensity. By modulated excitation of a long-wavelength Cy5 transient absorption, we dynamically modulate Cy5 emission. The frequency dependence enables specification of the dark-state timescales enabling optical-demodulation-based signal recovery from high background. These dual-laser illumination schemes for high-sensitivity fluorescence-signal recovery easily improve signal-to-noise ratios by well over an order of magnitude, largely by discrimination against background. Previously limited to very specialized dyes, our utilization of long-lived dark states in Cy5 enables selective detection of this very common single-molecule and bulk fluorophore. Although, in principle, the "dark state" can arise from any photoinduced process, we demonstrate that cis-trans photoisomerization, with its unique transient absorption and lifetime enables this sensitivity boosting, long-wavelength modulation to occur in Cy5. Such studies underscore the need for transient absorption studies on common fluorophores to extend the impact of fluorescence modulation for high-sensitivity fluorescence imaging in a much wider array of applications.
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute of Bioscience and Bioengineering, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
authors.forename: Chaoyang
authors.initials: C
authors.lastname: Fan
authors.forename: Jung-Cheng
authors.initials: JC
authors.lastname: Hsiang
authors.forename: Robert M
authors.initials: RM
authors.lastname: Dickson
mesh_terms: D002232:Carbocyanines / Q000737:chemistry*; D007536:Isomerism; D013050:Spectrometry, Fluorescence; D014966:Xanthenes / Q000737:chemistry
chemical_list: D002232:Carbocyanines; D014966:Xanthenes; C085321:cyanine dye 5; C034657:Texas red
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