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T issue Imaging with CARS Micr oscopy 323

arranged in a collinear geometry in this early nonlinear microscope.
Instead, Duncan et al. chose to adopt a noncollinear arrangement of
the beams. Such a geometry had become commonplace for CARS
measurements in condensed phase materials, which was motivated
by extending the interaction length over which the pump, Stokes and
anti-Stokes waves stay in phase during the signal generation process. 24
Indeed, noncollinear excitation geometries extended the range of phase
matching between the waves from less than a hundred micrometers
to up to several centimeters.
Although the anti-Stokes signal was generated from a relatively
large diameter focal spot, the spatial resolution of the first CARS
microscope was determined by the collection optics rather than the
focusing lens. The CARS light was captured by a microscope objec-
tive, filtered by a stack of spectral filters and projected onto a camera
placed in the image plane, yielding images with sub-micrometer res-
olution. The imaging speed of the CARS microscope lived up to the
expectations: within only 2 seconds, vibrationally sensitive images of
a 200 × 200 mm area at microscopic resolution were shot, clearly
claiming superiority over the much slower Raman microscope.
However, the spectral contrast was rather disappointing. Only
after use of image subtraction techniques could deuterated lipids in
dense liposomes clusters be discriminated from their nondeuterated
counterparts. 25,26 The gain in speed relative to the Raman microscope
was compromised by a significant loss in vibrational contrast, casting
some serious doubts on the practical benefits of CARS microscopy.

11.2.2 Second Generation CARS Microscopes
The low contrast in the first CARS microscopes was caused by the
presence of a strong nonresonant background. Scholten et al., who
27
built the first widefield CARS microscope, proposed several possi-
ble background rejection mechanisms, among which resonant
28
enhanced CARS and background cancellation by phase mismatching. 29
But was not until 1999, 18 years after the inception of CARS micros-
copy, that the technique was fortuitously resuscitated by Zumbusch
30
et al. and was saved from becoming a dust collecting curiosity. Key
to the success of the second generation of CARS microscopes was the
reintroduction of the collinear excitation geometry. Zumbusch et al.
realized that when the incident beams are focused by a high numeri-
cal aperture microscope objective, the interaction length between the
pump, Stokes and anti-Stokes fields, is so short that the waves are
unable to run out of phase. Hence, there is no need for a noncollinear
31
phase-matching arrangement of the beams per se. With the collinear
arrangement, smaller and cleaner focal volumes are produced, which
condenses the location of CARS signal generation to a sub-micrometer
spot in the sample. CARS generation in small, phase-matched vol-
umes has two important advantages: (1) the microscope has an
intrinsic three-dimensional resolution because of the confinement of