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 specificaton 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.

Tailoring Cyanine Dark States for Improved Optically Modulated Fluorescence Recovery

“…Excitation of cis-Cy5 in its absorption band, however, photoreverts the Cy5 to the trans state and recovers fluorescence with enhancements of up to 50%. 18 Although Cy5 excitation also shows triplet state dynamics for both trans- and cis-isomers, the T 1 levels have much shorter lifetimes of ~35 μs and 6 μs for the trans and cis isomers, respectively. 16 Thus, our approach of fluorescence excitation combined with secondary laser co-illumination that is on-resonance with the transient absorption dynamically modulates the Cy5 fluorescence intensity via optical depopulation of primarily the photoisomerized dark state.…”

Modulated Fluorophore Signal Recovery Buried within Tissue Mimicking Phantoms

“…The modulation frequency dependence has been shown to be indicative of the natural dark state lifetime. 20 Because the secondary laser was square wave modulated at 100 Hz, Cy5 fluorescence was also modulated at 100 Hz, as revealed by Fourier transformation of the fluorescence intensity (Figure 5A–B). As Texas red fluorescence is not modulatable, demodulated fluorescence signals are indicative of only Cy5 emission and are directly recoverable even when buried up to 6 mm deep within skin tissue phantoms (Figure 5B).…”

“…Secondary illumination, however, only depopulates the primary laser-prepared dark state of Cy5 without modulating Texas red emission. 20 Texas Red is more efficiently excited at 594nm than is Cy5, somewhat offsetting the better spectral match of the filter passband to the Cy5 emission. Regardless, ~25% of Texas Red and ~75% of Cy5 emission is collected in the collected spectral region.…”

“…Demodulation of collected fluorescence is directly performed at the externally applied modulation frequency, which is free of (unmodulated) background emission. 18–20 Crucial in heterogeneous biological systems, SAFIRe improves sensitivity primarily by removing obscuring heterogeneous background. Importantly, the low-energy secondary laser offers the opportunity to probe dyes more deeply embedded in tissue.…”

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery

“…We have shown that this amplitude scales linearly with the total number of modulatable molecules and is shifted to its own unique detection frequency, free from obscuring background signals. 9–10, 14, 21, 25 While this amplitude is linear in the number of molecules, the slope of Fourier amplitude vs. concentration of modulatable species is a product of many photophysical and experimental parameters. Thus, accurate numbers of molecules can only be obtained from the Fourier amplitude if calibrated by the correlation subtracted SAFIRe-FCS or through a predetermined calibration curve obtained under identical conditions.…”