Parametric study of the emission spectra and photobleaching time constants of a fluorescent dye in laser induced fluorescence photobleaching anemometer (LIFPA) applications

Abstract

As a novel velocity measurement technique for micro/nanofluidics applications, the laser-induced fluorescence photobleaching anemometer (LIFPA) has been proven to have simultaneously high spatial and temporal resolutions. Its temporal resolution, which is crucial for investigations of transient flows, is directly determined by the photobleaching time constant. In this study, the factors, e.g., fluorescent excitation and emission spectra and the photobleaching time constant that may affect LIFPA measurements, were investigated using the coumarin 102 fluorescent dye at different concentrations (10–300 μM) in a confocal-microscope-based system. We report that a blue shift of the emission spectrum in the range of 430–515 nm can be observed during photobleaching attributed to the photochemical reactions between coumarin 102 molecules. Meanwhile, different photobleaching time constants are also observed in different bands of the emission spectrum. All of the photobleaching time constants are below 3 μs, which is nearly an order smaller than the previously reported values. Among all of the spectral bands, the bands at 470–492 nm can provide a higher fluorescent intensity and better accuracy for velocity measurements. The bands at 430 and 460 nm can provide a higher temporal resolution.