In vivo hyperspectral CARS and FWM microscopy of carotenoid accumulation in H. Pluvialis
2014
Coherent anti-Stokes Raman scattering (CARS) and four-wave-mixing (FWM) microscopy are a related pair of
powerful nonlinear optical characterization tools. These techniques often yield strong signals from concentrated
samples, but because of their quadratic dependence on concentration, they are not typically employed for imaging or
identifying dilute cellular constituents. We report here that, depending on the excitation wavelengths employed, both
CARS and degenerate-FWM signals from carotenoid accumulations in alga cysts can be exceptionally large, allowing
for low-power imaging of astaxanthin (AXN) deposits in Haematococcus pluvialis microalga. By use of a broadband
laser pulse scheme for CARS and FWM, we are able to simultaneously collect strong intrinsic two-photon-excitation
fluorescence signals from cellular chlorophyll in vivo . We show that CARS signals from astaxanthin (AXN) samples in
vitro strictly follow the expected quadratic dependence on concentration, and we demonstrate the collection of wellresolved
CARS spectra in the fingerprint region with sensitivity below 2mM. We suggest that multimodal nonlinear
optical microscopy is sufficiently sensitive to AXN and chlorophyll concentrations that it will allow for non-invasive
monitoring of carotenogenesis in live H. pluvialis microalgae.
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