Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy

Multiplex coherent anti-Stokes Raman scattering (CARS) microscopic imaging is demonstrated for the first time and used to visualize the thermodynamic state, either liquid crystalline or gel phase, of lipid membranes. Whereas in spontaneous Raman scattering the low scattering cross section is generally prohibitive for high-resolution microscopic imaging, the signal enhancement in multiplex CARS, by more than 4 orders of magnitude, enables microscopic imaging with realistic frame rates (similar tomin(-1)). Because of the broad spectral window over which the CARS signal is detected simultaneously, the identification of the thermodynamic state of the lipid membranes can be accomplished directly, without any additional tuning of the lasers. We demonstrate that the experimental CARS spectral data are highly consistent with those obtained with spontaneous Raman scattering, with a signal-to-noise ratio determined only by Poisson noise. In addition, the inherent intensity normalization renders multiplex CARS a robust imaging technique.