Multiparameter label-free flow cytometry using multiplex coherent anti-Stokes Raman scattering (MCARS) with biological applications

Flow cytometry is an invaluable analytical tool that provides statistical information about sample populations. By optically probing samples at high-speed, flow cytometers retrieve information about basic morphology and composition. The optical elastic scatter is indicative of sample size and granularity while molecular information requires the addition of fluorescent labels, which have limitations such as spectral overlap, non-specific binding, and cellular toxicity. In this work, we present the first multiparameter label-free flow cytometer that observes the elastically forward-scattered light (FSC) and probes the intrinsic Raman vibrations of passing samples using multiplex coherent anti-Stokes Raman scattering (MCARS). MCARS, as a broadband technique, probes a large region of the Raman spectrum; thus, leading to rich molecularly-sensitive information. Additionally, we present the first experimental investigations of two biological systems using the multiparameter flow cytometer: Saccharomyces cerevisiae, a yeast often used as a model system for eukaryotic organisms, and Phaeodactylum tricornutum, a diatom under investigation for biofuel production.