Materials Science and Engineering Laboratory FY 2004 ... - NIST

Technical Highlights
Coherent Anti-Stokes Raman Scattering (CARS) for
In-Situ Chemical Imaging of Tissue Engineering Constructs
Design issues relating to bioactive devices for
regenerative medicine reflect the spatial and
chemical complexity of biological and materials
issues and their interactions. Tools developed
for the purpose of aiding understanding of these
systems must have sufficient discriminatory power
to sort out this complexity. We have introduced
a relatively simple broadband spectroscopic
microscopy based on CARS that can be used to
rapidly acquire volumetric, chemically-specific
images with submicrometer resolution.
Marcus T. Cicerone
Biological research in general could be significantly
aided by a high-resolution, chemically sensitive
volumetric imaging method that allows rapid, non-invasive
study of processes on the tissue, cellular and sub-cellular
levels. The need for such a method is particularly
acute in the field of tissue engineering, where cycles
of cell-seeding and analysis can take months. Standard
analysis methods are labor intensive and destructive,
so multiple endpoint studies must be performed and
temporal correlation assumed. Non-invasive microspectroscopic
imaging could alleviate many problems
associated with the evaluation of tissue scaffolds both
by allowing continuity of analysis on a single scaffold
construct, and by maintaining spatio-temporal information.
Our vision is the creation of a non-invasive microscopic
imaging technique that rapidly discriminates between an
arbitrary number of chemically distinct structures or
species in a spatially resolved way within a biological
system. We envision that this would be done without
staining or otherwise intrusively manipulating the system.
We expect that such a microscopy tool would have a
significant impact on the way in-vitro tissue-engineering
studies are carried out.
While our vision may sound fanciful, the basis
for such powerful and generally applicable chemical
discrimination was established many decades ago via
vibrational spectroscopy. Our contribution has been
to marry broadband vibrational spectroscopy, with
its inherent chemical resolving power, to a nonlinear,
multiphoton microscopy (CARS microscopy), which
has already been demonstrated to have significant utility
for non-invasively imaging biological systems, even in
the absence of significant chemical resolving power.
Microscopic coherent anti-Stokes Raman
scattering (µCARS) can answer important imaging
2
needs of the biological research community, including
relative non-invasiveness, rapidity, and chemical specificity.
CARS provides chemical specificity through its intrinsic
sensitivity to molecular vibrational transitions. CARS is
sensitive to the third order polarizability, χ (3) , which has
nonresonant and resonant components, the latter being
related to the Raman scattering cross section. Therefore,
µCARS uses Raman (vibrational) susceptibility as a
contrast mechanism, but it is approximately 10 3 more
efficient than spontaneous Raman scattering.
Figure 1: Energy level diagram for single frequency CARS
process (solid vertical arrows) and broadband CARS (solid
and dashed vertical arrows).
The solid vertical lines of Figure 1 represent
transitions made by molecules in the “classic” CARS
scheme. Molecules in a sample of interest are promoted
to a specific energy E = h (wp – wS), where ωp and ωS are the frequencies of the near infrared pump and
Stokes light pulses, respectively, and h is h/2π where h
is Planck’s constant. If that energy corresponds to a
particular excited vibrational state in the molecule, that
state is populated. A subsequent probe photon (also ωp) promotes the vibrationally excited molecule to another
(higher) virtual state, from which it relaxes and emits
an anti-Stokes photon at a specific frequency, ωaS. The flux of anti-Stokes photons detected is proportional
to the number of molecules initially prepared in the
targeted vibrationally excited state. To date, this
“classic” µCARS scheme has been used to acquire only
single-frequency or narrowband (200 cm –1 –
–
) vibrational
information, and thus, chemical specificity is limited
to only one or two species simultaneously.