KEYWORDS: In vivo imaging, Microscopy, Raman spectroscopy, CARS tomography, Absorption, Resonance enhancement, Microscopes, Signal processing, Visualization, In vitro testing
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.
Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy has provided an imaging tool for
extraction of 3-dimensional volumetric information, as well as chemically-sensitive spectral information. These
techniques have been used in a variety of different domains including biophysics, geology, and material science.
The measured CARS spectrum results from interference between the Raman response of the sample and a non-resonant
background. We have collected four dimensional data sets (three spatial dimensions, plus spectra)
and extracted Raman response from the CARS spectrum using a Kramers-Kronig transformation. However,
the three dimensional images formed by a CARS microscope are distorted by interference, some of which arises
because of the Gouy phase shift. This type of interference comes from the axial position of the Raman resonant
object in the laser focus. We studied how the Gouy phase manifests itself in the spectral domain by investigating
microscopic diamonds and nitrobenzene droplets in a CARS microscope. Through experimental results and
numerical calculation using finite-diference time-domain (FDTD) methods, we were able to demonstrate the
relationship between the spatial configuration of the sample and the CARS spectral response in three dimensional
space.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.