Unmixing algorithm in multiplex coherent anti-Stokes Raman scattering with application to label-free protein...

Coherent anti-Stokes Raman scattering (CARS) microspectroscopy has emerged as a powerful technique for probing molecular assemblies with high chemical specificity. However, differentiating proteins within the same family remains a significant challenge. Here, we address the problem of the discrimination between actin and myosin filaments in human muscle. Starting with an inverse problem formulation, we propose a two-step computational resolution. First, the pure CARS spectra of actin and myosin are estimated. Second, these spectra are used to perform a spectral unmixing onto the whole image in order to quantify the relative contribution of each protein at the pixel level. Experiments on human muscle samples demonstrate that automated, label-free, and qualitative discrimination between protein families can be achieved using CARS microscopy.