Page 350 - Vibrational Spectroscopic Imaging for Biomedical Applications

324 Cha pte r Ele v e n

the interaction volume in both the lateral and axial dimensions,
32
similar to the two-photon excited fluorescence microscope, and
(2) reduction of the focal volume assigns more importance to small
vibrationally resonant structures relative to the nonresonant signal
generated from the bulk. This latter notion is the major reason why
the nonresonant background in Duncan’s imaging system was
overwhelming whereas it is manageable in Zumbusch’s collinear
excitation microscope, where the focal volume is almost three
orders of magnitude smaller.
Although image acquisition times in the first incarnation of the
collinear CARS microscope were too long for practical biological
imaging, within 2 years subsequent improvements of the light source,
the scanning mechanism and the detectors brought the imaging rate
down to a couple of seconds per image. 33,34 Despite the fact that the
nonresonant background still imposed contrast challenges, for sev-
eral imaging applications, most notably the imaging of lipids, the
vibrational signal turned out to be strong enough to compose high-
contrast images. The first applications to visualizing intracellular
lipid bodies 35,36 and membranes 37–40 proved decidedly successful,
which spurred the continuously growing popularity of CARS micros-
copy as a useful visualization tool in cellular biology.
Since 1999, the field of CARS microscopy has grown beyond all
expectations. Active CARS research is conducted both on the techno-
logical front, which seeks to improve the chemical imaging capabili-
ties, and the applications front. Although CARS imaging has found
several applications in material science, 41–43 the majority of CARS
applications are within the realm of biological and biomedical
research. In this chapter, we will provide examples and prospects of
CARS in these latter areas. For a more extensive overview of the
developments in the CARS microscopy field since 1999, the reader is
referred to several excellent review papers in the literature. 44–47

11.3 CARS Basics
In order to better appreciate the imaging properties of the CARS
microscope, we will briefly explain the basic physical principles that
underlie this technique. Our discussion will be largely qualitative,
with an emphasis on the physical picture rather than their mathemat-
ical descriptions. The different principles on which the CARS technique
is based are not all conveniently explained within a single framework.
Some properties are easily explained within a classical mechanical
picture, whereas the clarification of other properties requires a quan-
tum mechanical framework. This dual picture is the source of some
confusion about some of the aspects of CARS. In the following, we will
exclusively adopt a classical picture to explain the generation of waves
at the anti-Stokes frequency. A similar discussion with more mathe-
matical representation can be found elsewhere. 48–50