Page 354 - Vibrational Spectroscopic Imaging for Biomedical Applications

328 Cha pte r Ele v e n

6
sensitive, with detection sensitivities of less than 10 CH modes in
2
focus at sub-ms pixel dwell times. 57
11.4.2 Photodamaging
Naturally, higher CARS signals are attained by increasing the
pulse power. However, photodamaging concerns put a practical
limit on how much power can be applied to the sample. Two types
of photodamage are relevant to this discussion. First, light absorp-
tion by components in biological materials, which scales linearly
with the average illumination power, produces heating of the sam-
ple. Using near-infrared radiation, sample heating is generally negli-
gible for average powers less than 10 mW in most biological
materials. 58,59 Second, nonlinear excitation of compounds in cells and
tissues may induce photochemical changes with possible toxic photo-
products, among which the formation of radicals. 58,60 In CARS stud-
ies, nonlinear photodamage is oftentimes the prime source of damage
61
to the sample. By keeping the pulse energies below 1 nJ, nonlinear
photodamage in CARS microscopy can be generally avoided for most
56
samples. In practice, imaging with focal intensities of 10 mW from a
~80 MHz pulse train produces excellent CARS signal levels while
photodamaging effects are kept to a minimum.

11.4.3 CARS Chemical Selectivity
The CARS imaging microscope has proven to be a very sensitive
tool to visualize the distribution of lipids in biological samples. For
instance, CARS has been used to follow the growth and trafficking
of lipid droplets in a variety of cell types 36,56 and microorganisms, 62
to visualize the agent-induced morphological changes to myelin
sheets in the spinal cord 63,64 and to map out lipid deposits in ath-
65
erosclerotic lesions. All these studies are facilitated by the high
density of CH modes in lipids, which produces a CARS strong sig-
2
−1
nal at its symmetric stretch vibration at 2845 cm . As illustrated by
Fig. 11.3, the lipid CARS response also benefits from having its
major signatures in a relatively quiet region of the vibrational spec-
trum, which prevents spectral interferences with neighboring
bands. Other dense CH -containing compounds and a concentrated
2
substance like water can also be relatively easy visualized in the
−1
high-frequency range (2500 to 3500 cm ) of the vibrational spec-
trum. Specificity among lipids and other CH -containing com-
2
pounds can furthermore by obtained through the use of deuterium
labels. 25,57,66,67
−1
The situation in the fingerprint region (~800 to 1800 cm ) is,
however, quite different. Unlike the limited number of molecular
modes in the high-frequency region, many molecular groups have
their frequencies in the fingerprint region. Indeed, a typical Raman
spectrum from a biological sample is characterized multiple partially