Page 359 - Vibrational Spectroscopic Imaging for Biomedical Applications

T issue Imaging with CARS Micr oscopy 333

simultaneously. Indeed, in one of the first rapid CARS imaging stud-
ies of living cells, the CARS technique was combined with simultane-
ous two-photon-excited fluorescence lifetime microscopy (FLIM). 34
Other examples include multimodal imaging based on CARS, TPEF,
SHG, and sum frequency generation (SFG) microscopy. 65,84
The CARS imaging microscope enables a complete multimodal
investigation of tissues based on endogenous contrast. In addition to
the chemical selectivity offered by CARS, simultaneously detected
SHG signals reveal tissue collagen patterns and TPEF signatures
report on endogenous fluorophores such as elastin fibers and nicotin-
amide adenine dinucleotide (NAD) metabolic agents. 85,86 The CARS
microscope thus constitutes the ultimate nonlinear imaging platform.

11.6 CARS in Tissues
The major difference between thin samples (μm sized) and tissues is
the presence of significant light scattering in the latter. Light scatter-
ing is a consequence of variations in the linear refractive index in the
sample. In tissues, refractive index variations result from large struc-
tures such as extracellular fibers and smaller structures such as intra-
cellular organelles in an otherwise aqueous environment. In addition,
light absorption is also relevant for thicker tissues. Both light scatter-
ing and absorption give rise to signal loss in tissues relative to thin
samples. The consequences of these linear optical effects on the CARS
signal will be discussed below.

11.6.1 Focusing in Tissues
Optimum CARS signals are obtained when the incoming light is con-
densed into a tight focal spot. Naturally, light scattering in tissues
compromises the formation of a clean focal spot. The reason for this
is twofold. First, scattering of excitation light in the tissue leads to loss
of amplitude in the vicinity of the focal volume. 87–89 Second, the phase
of the incident waves will be compromised upon arrival in the focal
region. The focal volume, which exists by virtue of constructive inter-
90
ference of light waves in one point in space, is very sensitive to the
coherence of the incoming light. Loss of phase coherence implies that
the waves will be unable to completely interfere to produce a tight
focal spot. Hence, in the presence of scattering, the focal volume will
be smeared out and contain a lower excitation density. CARS signals
are directly affected and generally much lower if tissue scattering is
significant.
Using NIR radiation, appreciable CARS signals up to 0.25 mm can
generally be produced in most tissue types. Special long working dis-
tance objectives can often be used to increase this penetration depth by
a factor of two. Beyond 0.5 mm, light scattering severely complicates
the formation of a sufficiently tight focal spot for CARS generation. To