Biomedical Engineering Reference
In-Depth Information
of nonlinear optical phenomena such as multiphoton fluorescence, harmonic
generation, sum-frequency generation, coherent Raman scattering, and parametric
oscillation. In modern imaging systems, these optical effects are frequently used to
visualize the biological material from cellular to molecular level.
Multiphoton excitation process was first studied by Maria Goeppert-Mayer [
1
]
in 1931, but multiphoton microscopy was not developed until decades later. The
technology was patented by Winfried Denk, James Strickler, and Watt Webb in
1991 [
2
]. The first multiphoton optical process used in microscopy was second
harmonic generation (SHG) [
3
,
4
] that was followed by coherent anti-Stokes Raman
spectroscopy (CARS) [
5
]. Two-photon excitation fluorescence (TPEF) microscope
for biological imaging was initially demonstrated by Denk et al. [
6
]. Later, third
harmonic generation (THG) mode of multiphoton effect was used in a microscope
by Barad et al. in 1997 [
7
]. Since these earlier works, multiphoton imaging has been
applied in various modes of imaging in biomedical fields.
Multiphoton imaging is based on nonlinear optical response of a medium,
that is, optical processes that involve more than one photon interacting simul-
taneously with a molecule. Such interactions of photons with a material result
in either two or more photon absorption or scattering, as in two- and three-
photon excited fluorescence, second and third harmonic generation effects, and
coherent anti-Stokes Raman scattering. Multiphoton imaging can be divided into
incoherent and coherent modes of a nonlinear optical microscope. The signal in
incoherent multiphoton imaging is characterized by emission of fluorescence or
phosphorescence having a random phase and whose power is proportional to the
concentration of radiating molecules. The principle of incoherent imaging system is
based on simultaneous absorption of two or more photons, as in, two-photon excited
fluorescence microscopy [
6
] and three-photon excitation fluorescence microscopy
[
8
,
9
]. The coherent multiphoton imaging is characterized by the emission of light
that is in phase with the excitation field and whose power is proportional to the
geometrical distribution of radiating molecules. The coherent multiphoton imaging
techniques are SHG microscopy [
10
,
11
], CARS microscopy [
5
,
12
], and THG
microscopy [
7
,
13
-
15
].
Two-photon excited fluorescence is the most commonly used multiphoton effect
in optical microscopy and has become an invaluable tool for biological imaging.
Two photons from a pulsed laser are simultaneously absorbed by a fluorophore,
and a single photon is emitted in the form of fluorescence that can be used to
make an image. TPEF can be used in materials with endogenous fluorophores or
with labeling by exogenous dyes or quantum dots. TPEF has the advantage of
multicolor labeling of various tissue elements and subcellular organelles within a
single biological sample. Like all contrast methods based on fluorescence, TPEF
also suffers from photobleaching and photodamage. Second harmonic generation,
on the other hand, is a nonlinear optical process that is biologically compatible,
noninvasive, and requires no labeling.
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