Multiplex CARS microspectroscopy in the “long-pulse” regime: where are we now?

Since its first lab demonstration in 2008, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the “long-pulse” regime (50 ps - 1 ns) has become a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. In this paper, we review the last developments relative to this technology, in terms of instrumentation (simplified MCARS), data analysis (unsupervised chemical analysis of hyperspectral big data) and biological applications (cell/tissue imaging, time-lapse imaging). It is reminded that running MCARS microspectroscopy in such long-pulse regime allows to get the temporal overlapping of the pump and all the Stokes spectral components without dispersion compensation, enabling simultaneous and effortless hyperspectral operation in both O-H, C-H and fingerprint regions. This simplification of the experimental setup is consolidated by the use of a dual-fiber-output laser source, for which the synchronization between the pump and Stokes pulses can be adjusted by equalizing the fiber lengths of both arms, without the need for a delay line. Chemometric methods as multivariate curve resolution (MCR) are most appropriate for the unsupervised analysis of MCARS hyperspectral data. MCR is an iterative matrix decomposition method, constructing an approximation of data by means of their projection into a subspace guided by different constraints. In this context, we introduce a new approach of cell/tissue imaging, based on a simple workflow and without any phase retrieval computation.