In vivo hyperspectral CARS and FWM microscopy of carotenoid accumulation in H. Pluvialis

Aaron D. Slepkov; Aaron M. Barlow; Andrew Ridsdale; Patrick J. McGinn; Albert Stolow

doi:10.1117/12.2038229

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17 February 2014 In vivo hyperspectral CARS and FWM microscopy of carotenoid accumulation in H. Pluvialis

Aaron D. Slepkov, Aaron M. Barlow, Andrew Ridsdale, Patrick J. McGinn, Albert Stolow

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Proceedings Volume 8937, Multimodal Biomedical Imaging IX; 893709 (2014) https://doi.org/10.1117/12.2038229
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

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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Aaron D. Slepkov, Aaron M. Barlow, Andrew Ridsdale, Patrick J. McGinn, and Albert Stolow "In vivo hyperspectral CARS and FWM microscopy of carotenoid accumulation in H. Pluvialis", Proc. SPIE 8937, Multimodal Biomedical Imaging IX, 893709 (17 February 2014); https://doi.org/10.1117/12.2038229

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