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.