A key application of multiphoton microscopy has been for noninvasive imaging
deep within scattering media, such as biological tissue. Because multiphoton
microscopy falls within the broaderfield ofnonlinear opticsornonlinear optical
microscopy, it also provides several contrast mechanisms [ 24 ]. These mechanisms
include two-photon excitationfluorescence (TPEF), second-harmonic generation
(SHG), third-harmonic generation (THG), sum-frequency generation (SFG), stim-
ulated Raman scattering (SRS), and coherent anti-Stokes Raman spectroscopy
(CARS). More details on CARS and SRS are given in Chap. 9. These contrast
modalities enable the extraction of information about the structure and function of
the specimen under consideration, which is not available in other optical imaging
techniques.
8.6 Raman Microscopy
As described in Chap. 6 , Raman scattering occurs when photons undergo an
inelastic scattering process with a molecule [ 26 – 28 ].Raman microscopyis the
combination of Raman scattering with optical microscopy. This is a widely used
analysis technique for biological specificity (which describes the selective attach-
ment or influence of one substance on another, for example, the interaction between
an antibody and its specific antigen) and provides lateral resolution down to the
subcellular level. The detection and analysis of this inelastically scattered light is
the key function in Raman spectroscopy and is used to obtain information about
molecular compositions, structures, and interactions in biological tissue samples.
When monochromatic laser light impinges on a specimen sample that is being
examined, in a Raman inelastic scattering event a small amount of the incident light
(a fraction of about 10−^6 ) interacts with molecular vibrations in the sample and is
scattered at a slightly different wavelength. That is, there is an energy shift between
the excitation light and the Raman-scattered photons. In this process, either a small
amount of energy is transferred from the photon to the vibrational modes of the
molecule (calledStokes scattering), or the molecular vibrations can transfer some
energy to the photon (calledanti-Stokes scattering). Thus, as shown in Fig.8.15,
for Stokes scattering the deflected photon has a lower energy (longer wavelength)
than the incident photon, whereas for anti-Stokes scattering the deflected photon
has a higher energy (shorter wavelength) than the incident photon. This effect is the
basis of inelastic light scattering. Because the energy shift is a function of the mass
of the involved atoms and the molecular binding strength, every molecular com-
pound has a unique Raman spectrum. Thus, a plot of the intensity of the inelasti-
cally scattered light versus its frequency can be used to identify the sample. More
details on advanced setups and applications are given in Chap. 9 for Raman
spectroscopy, surface enhanced Raman scattering (SERS) spectroscopy, coherent
anti-Stokes Raman scattering (CARS) spectroscopy, and stimulated Raman scat-
tering (SRS) spectroscopy.
8.5 Multiphoton Microscopy 253