Development of a Fluorescence Lifetime Spectroscopy System with Multicolor-Excitation

Abstract

Fluorescence lifetime spectroscopy is an essential tool in various diagnostic applications, owing to its excellent sensitivity, concentration independence, and non-invasive capabilities. This method requires precise timing electronics capable of resolving time delays at the picosecond scale. However, commercially available systems suffer from issues such as bulkiness, high cost, and suboptimal performance. In this work, we introduce a cost-effective and compact system for measuring the fluorescence spectrum and fluorescence lifetime from samples in a cuvette, featuring a user-friendly graphical user interface (GUI). We report a timing resolution of 55 ps, with a full detection range of 500 ns, suitable for a vast majority of fluorophores. Specifically, the timing module is configured in reverse mode to reduce the effect from low photon count rate. When integrated with an additional supercontinuum excitation, the combined system enables a versatile excitation wavelength tuning within the range of 400–700 nm, with a minimal full width half maximum (FWHM) of the instrument response function (IRF) at 520 ps. To validate the system performance, we measure the concentration-dependence absorption and emission spectra, along with the fluorescence lifetime, of commercially available Cy5 and Fluorescein reference dyes, from which the results are consistent with previously reported data. Our systems should be of interest for various researchers interested in measuring multi-modal fluorescence properties.