Multimodal Biomedical Imaging Systems - Biomedical Optical Imaging Technologies: Design and Applications - page 298

9.1.7 Multiphoton Imaging

Multiphoton imaging uses pulsed long-wavelength light to excite fluorescence at
shorter wavelengths. A fluorophore absorbs the energy from two or more photons
with long wavelength simultaneously; the summed energies of long wavelength
exciting photons will produce an emission wavelength shorter than the excitation
wavelength. The fluorescence signal can be generated from exogenous probes
applied to the tissue or endogenous molecules that are inherently expressed.
Multiphoton imaging has attractive advantages over confocal imaging for live
cells and for tissues with three-dimensionally resolved fluorescence imaging.
Multiphoton excitation occurs only at the focal point of the objective lens, therefore
minimizing the photobleaching and photodamage that are limiting factors in
imaging live cells. Also, because the excitation wavelength is longer, multiphoton
imaging provides superior optical sectioning at greater depths in thick specimens
than is possible by other methods. Long wavelength and low-energy excitation lasers
also cause less damage than short-wavelength lasers so that cells may be observed
for longer periods with fewer toxic effects.
Similar to confocal imaging, multiphoton imaging has the limitations of small
FOV and low image acquisition speed. Another drawback is the cost of ultrafast
lasers used in multiphoton imaging.

9.1.8 Spectroscopy

Spectroscopy obtains detailed information about wavelength-dependent optical
properties of tissue through the emitted or absorbed spectrum. Depending on the
nature of the light and tissue interaction, several spectroscopy systems, such as
scattering spectroscopy, fluorescence spectroscopy, and Raman spectroscopy, have
been developed.
Scattering spectroscopy measures the amount of light that a substance scatters
at certain wavelengths, incident angles, and polarization angles. There are several
scattering spectroscopic technologies, such as diffuse reflectance spectroscopy
(DRS), elastic scattering spectroscopy (ESS), light scattering spectroscopy (LSS),
backscattering spectroscopy, and Raman spectroscopy.
Fluorescence spectroscopy measures autofluorescence or the exogenous fluores-
cence spectrum of the tissue. It can probe tissue to depths of up to several hundreds
of microns and reveal intrinsic signatures that can be correlated to microstructures
and biochemical composition of epithelial tissues.
Raman spectroscopy is a powerful method for molecular detection and charac-
terization; it is particularly attractive for biological and biomedicine applications
because it provides molecular-level information without the need to use exogenous
fluorescent labels or the need to perturb the sample using stains or chemical fixa-
tives. It can provide valuable information on chemical and structural changes due to
disease as well as mechanical deformation induced by aging or a prosthetic implant.