Two-photon excitation microscopy is typically associated with the following characteristics: (1) the wavelength for two-photon absorption is typically (though not always) twice the wavelength for single-photon excitation; (2) the penetration depth of the excitation light is considerably greater since the longer-wavelength light shows less scatter and therefore can penetrate deeper into highly scattering specimens; (3) the longer-wavelength near-infrared light is less damaging to live cells and tissues; however, specimens with high absorption coefficients in the infrared can still show thermal damage; (4) the axial optical sectioning capability is intrinsic to the physics of the two-photon absorption process; hence, confocal pin-holes are not required; (5) descanning of the emission is not required; and (6) a high SNR can be achieved since the excitation and emission wavelengths are widely separated.
As shown in the foregoing chapter, the two-photon process occurs through a virtual intermediate state. What is not mentioned is the process in which the first photon excites the molecule into a real, intermediate state, and the second photon excites the molecule from the intermediate state to the final excited state. This process is resonant two-photon excitation.
The physics of the two-photon excitation process leads to some extremely useful consequences. The probability of the electronic transition depends on the square of the instantaneous light intensity; this quadratic dependence follows from the requirement that the fluorophore must simultaneously (within the lifetime of the virtual state) absorb two photons per excitation process.
The laser light in a two-photon excitation microscope is focused by the microscope objective to a focal volume. Only in this volume is there sufficient intensity to generate appreciable excitation. The low photon flux outside the volume results in a negligible amount of fluorescence signal. In summary, the optical sectioning (depth discrimination) capability of a two-photon excitation microscope originates from the nonlinear quadratic dependence of the excitation process and the strong focusing capability of the microscope objective.
Most biological specimens are relatively transparent to near-infrared light. The focusing of the microscope objective results in two-photon excitation of ultraviolet-absorbing fluorochromes in a small focal volume. It is possible to move the focused volume through the thickness of the sample and thus achieve optical sectioning in three dimensions.
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