Development of Far-Red and Ultraviolet Digital Frequency-Domain Flow Cytometry Systems

Abstract

Flow cytometry is a powerful single cell analysis technique that uses fluorescence and light scatter to characterize differences (i.e., phenotype, genotype, morphology, etc.) in large cell populations. Digital excited state lifetime measurements with flow cytometry have been largely underutilized; the measurement of fluorescence decay rates by molecules in or on the surface of cells is challenging to accurately measure when the samples are moving rapidly through the path of a laser excitation source. In past work, we have shown that time-resolved cytometry can provide accurate measurements of the fluorescence lifetime of organic fluorophores at standard cytometric throughputs (∼1000 cells/s). Herein, we advance our digital laser modulation and signal processing approaches to develop cytometry systems that operate across a broader spectral range. We optimized a far-red and ultraviolet (UV) cytometry instruments and present fluorescence microsphere results to determine detection sensitivity. The expansion of time-resolved systems across a wider color spectrum is important for many new applications including the development of near-infrared fluorescent proteins and the detection of UV-excitable autofluorescence in single cells.