Biology Reference
In-Depth Information
major drawbacks of confocal microscopy such as the restricted penetration depth
and photodamage, is o
V
ered by multiphoton microscopy systems.
XI. Multiphoton Excitation Laser Scanning Microscopy
erent microscope that also allows optical sectioning and high
spatial and temporal resolution live specimen imaging, and that also comes with
other added benefits, is the multiphoton microscope. From a biophysical perspec-
tive, multiphoton imaging rests on an excitation principle that relies on excitation
of the fluorescent molecule by photons that alone do not have enough energy, but
need to combine by simultaneously coming in to a very close proximity of the
fluorophore.
Although the theoretical concept of multiphoton excitation is relatively simple and
has been known and also utilized by physicists for many decades, biological applica-
tions of multiphoton excitation are of amore recent date (
Denk et al.,1990
).Whereas
in confocal single-photon laser illumination, the fluorophore is excited by the absorp-
tion of a single photon, as it provides su
A conceptually di
V
cient energy for the fluorophore to reach an
excited state. Upon return to the ground state, a photon of longer wavelength than
the excitation photon is emitted, and it is this process that creates the fluorescence.
With multiphoton excitation, a fluorophore is excited by the near-simultaneous
(within 10
18
s) absorption of two or more photons that combined provide enough
energy to promote the fluorescent molecule from a ground state to an excited state;
two photons for two-photon (2P) excitation, three photons for three-photon (3P)
excitation, etc., with 2P excitation being by far the most common multiphoton
excitation modality. Thus, with 2P excitation, the fluorophore absorbs two photons
simultaneously, each twice the wavelength and half the energy required for molecular
excitation, and likewise, in 3P excitation, each of the three excitation photons have
three times the wavelength, but only one-third of the energy compared to single-
photon excitation (
Helmchen and Denk, 2005
).
Once excited, the emitted fluorescence is then proportional to the square of the
excitation intensity in 2P absorption (third power in 3P excitation), and this 2P
excitation (measured in Goeppert-Meyer Units) occurs only at the focal point, as
it is only here that the density of the excitation photons is high enough to ensure
a simultaneous photon arrival to the fluorophore that is su
Y
cient to excite it.
Though this may not occur with all photons in the volume, the probability of it
occurring for the vast majority of the photons is very high. This nonlinear
excitation constitutes the most important physical di
Y
erence from confocal sin-
gle-photon excitation, in that excitation will be confined to a small ellipsoid
volume around the focal point, whereas above and below this point, the density
of photons su
V
ciently close to one another, or in other words the light intensity,
will not be high enough to generate any excitation (
Fig. 6
). This e
Y
ectively means
that out-of-focus fluorescence will not be generated, which again removes the
need for a confocal aperture with a pinhole in the emission light pathway.
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