Multiplex coherent anti-Stokes Raman scattering flow cytometry for real-time classification of particles in a microfluidic channel

Flow cytometry is an ever-advancing, multivariate analysis tool obtaining morphological and phenotype information from sample populations. In traditional flow cytometers, obtaining molecular information requires the use of endogenous fluorophores, which are limited by spectral overlap, nonspecific binding, available conjugation chemistries, and cellular toxicity. In this work, however, we apply multiplex coherent anti-Stokes Raman scattering (MCARS), a nonlinear optical method that probes the Raman energies within a molecule, to flow cytometry in order to demonstrate label-free, molecularly sensitive particle differentiation. To demonstrate this, we correctly differentiate 5 μm polystyrene (PS) and poly(methyl methacrylate) (PMMA), which (linearly) optically appear similar, but vary significantly in their MCARS spectrum.