Create and organize publications into your own personal collections/lists
Easily search saved publications across your mulitple lists
Share your collections with friends, coworkers, or anyone that might be interested in the same research
To take advantage of My Library, sign in now.
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.
Online access to SPIE eBooks is limited to subscribing institutions.