Sub-mM imaging of carotenoids using electronic and vibrational nonlinear optical microscopy

Carotenoids are highly conjugated natural polyenic compounds with notoriously large optical nonlinearities. The commercial importance of carotenoids such as β-carotene and astaxanthin currently exceeds $500M, as these compounds are used as dyes, natural supplements, cosmetics, and food additives. Furthermore, biologically, carotenoids are used for many purposes, including as optically important components in photosynthis complexes. The large optical nonlinearities in carotenoids arises from the long conjugated backbone of the carotenoid molecule. Vibronic coupling between the highlydelocalized electron modes along the backbone and certain vibrational modes yields exceedingly large Raman and stimulated Raman responses, even at wavelengths far removed from traditional "resonance Raman" conditions. We report on the exploitation of such large natural nonlinear vibrational responses for enabling both coherent Anti-Stokes Raman scattering (CARS) and electronic four-wave-mixing (FWM) microscopy of dilute concentrations of carotenoids in vitro and in live microalgae. We find that CARS/FWM signals from astaxanthin in pump wavelength regimes between 800nm-920nm surpasses that from bulk diamond by orders of magnitude, allowing for nearly-nonresonant-background-free hyperspectral CARS imaging down to below 1mM. These results strongly support the potential for CARS/FWM to be used for live monitoring of carotenogenesis in plants and microorganisms, and thus may also apply to the study of natural and artificial photosynthesis.