Lynn Savage, email@example.com
GAITHERSBURG, Md. – A refinement of coherent anti-Stokes Raman spectroscopy engineered by
researchers at the National Institute of Standards and Technology (NIST) has resulted
in a speedy and powerful new technique for label-free, chemically sensitive analysis
of cells and their components.
A broadband version of coherent anti-Stokes Raman spectroscopy
(B-CARS) provides the same amount of chemical contrast available when looking at
cells with spontaneous Raman, but does so at a much faster rate, according to the
A new Raman microspectroscopy technique called broadband CARS provides
chemical fingerprinting information and C-H stretch signals. Bright-field microscopy
images of mouse cells (left) are contrasted with an image generated with B-CARS
data (right). Green shows the cell’s nucleus; blue is the cytosol. Courtesy
Spontaneous Raman microspectroscopy techniques can help elucidate
the morphology and chemical composition of subcellular components in biological
tissues and cells, but sample preparation constraints and long spectral acquisition
times make it difficult to use.
B-CARS can accomplish the same spatial resolution and chemical
discrimination, but with relaxed sample constraints and at a much higher spectral
acquisition rate, because it uses multiphoton excitation to generate a coherent
Because the signal is anti-Stokes, intrinsic fluorescence that
plagues spontaneous Raman in biological samples is not an issue for B-CARS. And
because the B-CARS signal is coherent, intrinsic heterodyning can be used to boost
the weak Raman signal. Using this intrinsic heterodyne amplification, the NIST team
has achieved signal-to-noise ratios of 10:1 in even the weak fingerprint spectral
regions, with only a 50-ms spectral acquisition.
“We have already reduced the spectral acquisition time from
seconds (as with spontaneous Raman) down to tens of milliseconds; if we can push
to submilliseconds, we could really open up Raman as a first-line investigative
tool for biology,” said Marcus T. Cicerone, a project leader in the biomaterials
group at NIST. Cicerone began development of the technique several years ago, after
seeing results from the narrowband CARS efforts of Xiaoliang S. Xie’s lab
at Harvard University.
Most CARS-based microscopy is performed at a single frequency,
typically in the C-H stretch region between 2840 and 3000 cm—1. Although sufficient
for high-speed analysis of some cellular structures, single-frequency imaging does
not pick out the more subtle chemical differences that could help with disease diagnoses
or other important traits.
B-CARS, however, simultaneously acquires vibrational spectra between
500 and 3200 cm—1, at each pixel of a target, unveiling both the chemical fingerprint
of each component as well as its C-H stretch signals.
“Narrowband CARS is much faster – with microsecond
acquisition – but chemical specificity is always a question,” Cicerone
said. “You never really know if your (Raman) signal is coming primarily from
C-H stretches or just changes in the nonresonant susceptibility of the sample. For
purely morphological imaging, this may not matter, but if one wants to do chemical
imaging, you really need some spectral bandwidth.”
The B-CARS system includes a Spectra-Physics 830-nm Mai Tai laser,
an Olympus IX-71 microscope and an Andor Technology BRDD-920 CCD camera. Pulses
from the laser were split into two: an 850- to 1200-nm Stokes pulse created within
a photonic crystal fiber supplied by NKT Photonics and a narrowband probe pulse
centered at 830 nm. Just as important, however, is the analytical program Cicerone’s
The researchers’ system generates significant nonresonant
background alongside the resonant Raman peak signals within the spectrum. This nonresonant
component is typically viewed as a nuisance, but the NIST team used that nonresonant
signal to amplify the weaker fingerprint signal via heterodyning.
“We extract the resonant signal of interest with a modified
Kramers-Kronig transform algorithm that we invented to handle the nonresonant component
of the CARS signal,” Cicerone said.
The group is working on methods to improve the system’s
spectral acquisition speed and simplicity of use, with an eye toward making it
sufficiently user-friendly for biotech labs and clinical settings. Several new laser
and detector technologies may help to make this possible, Cicerone said.