Multiphoton
Excitation Principles
In MPE,
ultrafast 100 femtosecond pulsed lasers, when focused, produce
such high peak photon densities that simultaneous absorption of
two or more photons can occur. Each multiple absorption induces
a molecular excitation of a magnitude equivalent to the sum of
the absorbed photon energies. The simplified molecular energy
level (Jablonski)
diagram below shows that two or three red photons interacting
simultaneously with a molecule have a combined energy appropriate
to excite a deep UV electronic transition within the molecule.
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MPE excitation
is a nonlinear process that provides a unique localization to the
beam focus. Only at the focus are photon flux densities sufficient
for multiple photons to arrive “simultaneously” (in 10–15
seconds) at an excitable molecule (of 10–16 cm2
cross section). The second figure shows a photograph of the fluorescence
excitation profile produced by a laser beam focused into a fluorescent
solution. With a one-photon source (below) excitation events occur
throughout the beam profile, (b) With a two-photon source (above),
excitations are limited to the beam focus. Focal point restriction
of excitation provides intrinsically 3-d resolved submicron information
within thick specimens. MPE microscopy exhibits several advantages
over confocal microscopy:
- Photodamage
is restricted to the focal plane.
- In laser
scanning microscopy it is not necessary to refocus the descanned
fluorescence through a confocal aperture in the detector plane.
Instead, detection optics can be simple and efficient and the
scattering of light by thick, cloudy specimens does not interfere
with image formation, allowing 2-3 fold deeper penetration into
tissue.
- UV absorbing
and deep UV absorbing molecules are easily accessed with practical
(visible and near IR) wavelength ranges with no out-of-focus photodamage.
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| Photo:
Brad
Amos, MRC, Cambridge, UK |
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