A Novel Microfluidic-Based Fluorescence Detection Method Reveals Heavy Atom Effects on Photophysics of Fluorophores With High Triplet Quantum Yield: A Numerical Simulation Study

The present study introduces the idea of a novel fluorescence-based imaging technique combined with a microfluidic platform that enables a precise control of dark transient state populations of fluorescent probes flowing over a uniform, top flat supergaussian excitation field with a constant flow rate. To demonstrate the imaging capability of the proposed detection method, numerical simulations have been performed by considering laser, microscope and flow parameters of experimental setup together with photophysical model and electronic transition rates of fluorescent dyes. As an output data to be assessed, fluorescence image data is simulated numerically for bromine-free carboxyfluorescein and its brominated derivatives having different numbers of bromine atoms. Based on the magnitudes of applied excitation irradiances and flow rates, which can be manually controlled by user during experiments, the presence of dark state populations can appear as broadening, shifts and decays in normalized fluorescence intensity signals that are computed from simulated fluorescence images. As such changes in signals become more pronounced upon an increase in the degree of bromination, it is elicited that heavy atom effect can be resolved by properly tuning excitation powers of laser and flow rates. Proposed imaging method has potential to provide invaluable means to conventional fluorescence methods and can open up new perspectives in biomedical research.