Multi-Photon Excitation
Multi-photon excitation is a process in which an atom or molecule is excited by the absorption of multiple photons instead of a single photon. A simplified cartoon representation of this process can be seen below.
The figure shows the absorption of one, two, and three photons to an excited state Si from the ground state, followed by a non radiative transition to an intermediate state S1 before transitioning back to the ground state and emitting a photon in the process. This figure shows the process as one where the multiple photon absorption involves photons of one half or one third of the energy, per photon, required to make the transition to the excited state. The two or more photons must arrive within a time span on the order of a single femtosecond (10-15 s), thus requiring very high photon flux.
The images below show scanning fluorescence images of 4 different fluorophores, each imaged with the same excitation source: a 50 fs pulsed ti:sapphire laser with a center wavelength of 830 nm with a full width half max of 14 nm.
You may notice that none of the absorption maxima are at wavelengths of half that of the excitation source. This suggests that the above cartoon may need some modification. Indeed, a more accurate version involves the excitation to a quasi-state, beyond the excited state Si, from which it undergoes non-radiative relaxation to the excited state. A modified excitation-radiation scheme is shown below.
The excitation of fluorophores to a quasi-state allows for excitation of multiple fluorophores using the same excitation source providing a major benefit in comparison to single photon excitation. The images below show simultaneous excitation of three fluorophores, each one acting as a label for a different cellular structure.
In addition to the added benefit of simultaneously imaging dyes with different absorption characteristics, multi-photon fluorescence microscopy also reduces the need for spatial filtering of the fluorescence signal. Traditional confocal fluorescence microscopy requires the use of an aperture in the image plain of the microscope in order to spatially discriminate the signal to reduce background because of the large excitation volume. By contrast, multi-photon excitation has an inherently small focal volume due to the requirement of high photon densities. A comparison of excitation volumes between single and multi-photon is shown below.
The figure above shows a comparison between single photon (left) and multi-photon (right) excitation volumes, shown in green. Here you see the excitation volume is restricted to the focal volume leading to the inherent spatial discrimination along the direction of excitation source’s propagation. This is what allows for the reduction or elimination of the need for an aperture in the image plane for noise reduction as only those fluorophores within the small excitation volume.
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