Two-photon and three-photon excitation occurs as the result of simultaneous fluorophore absorption by either two or three photons in a single quantitized event. This tutorial explores how fluorescence excitation events occur in multiphoton microscopy utilizing the classical Jablonski diagram.

The tutorial initializes with the wavelength selection guide (left-hand side of the window) set to 700 nanometers, on the border between the visible and near-infrared range. This wavelength is commonly utilized for two-photon excitation with Ti:sapphire pulsed mode-locked laser systems. The excitation mode is dictated by the wavelength marker on the wavelength selection guide. Between 300 and 600 nanometers, the tutorial is in single-photon excitation mode, and clicking on the Start button will launch a single electron to an excited state after the photon has arrived. the excitation and emission wavelengths, and their associated colors, are presented on the right-hand side of the tutorial window. When the marker is set between 600 and 1000 nanometers, the tutorial is in two-photon excitation mode. Clicking on the Start button will launch two photons whose arrival time is dictated by the Excitation Delay slider. When the slider is in the range 10 e(-18) to 10 e(-20), the two photons will arrive in time for the electron to be promoted through the virtual state to an excited singlet energy state. Longer times will not allow the transition. Adjusting the wavelength marker to values higher than 1000 nanometers will place the tutorial in three-photon excitation mode. In this mode, the tutorial behaves in a similar manner to the two-photon state, except that three photons must be incident for excitation to occur.

At high photon densities, two photons can be simultaneously absorbed (mediated by a virtual state) by combining their energies to provoke the electronic transition of a fluorophore to the excited state. Because the energy of a photon is inversely proportional to its wavelength, the two photons should have wavelengths about twice that required for single-photon excitation. As an example, two photons having a wavelength of 640 nanometers (red light) can combine to excite an ultraviolet-absorbing fluorophore in the 320-nanometer region (ultraviolet), which will result in secondary fluorescence emission of longer (blue or green) wavelengths. This unique application means that longer wavelengths, extending into the infrared region, can be conveniently utilized to excite chromophores in a single quantum event, which subsequently emit secondary radiation at lower wavelengths.

The requirement of two photons for each excitation event necessitates a rate constant that depends upon the square of the excitation intensity. Although the photons do not have to be of identical wavelength to induce multiphoton excitation, most experimental systems are designed with a single laser source, so the two photons are usually members of a defined population having a narrow wavelength distribution. Unlike the case for single-photon absorption, the probability that a given fluorophore will simultaneously absorb two photons is a function of both the spatial and temporal overlap between the incident photons. Calculations based on the assumption that each fluorophore is exposed to the same laser cross section indicate that photons must arrive within 10(-18) seconds (one attosecond) of each other. The time scale of this overlap period is consistent with the lifetime (10(-17) seconds or 0.01 femtosecond) of the intermediate virtual state.

Three-photon excitation is a related non-linear optical absorption event that can occur in a manner similar to two-photon excitation. The difference is that three photons must interact simultaneously with the fluorophore to elicit a transition to the excited singlet state. A benefit of three-photon excitation is that successful absorption requires only a tenfold greater concentration of photons than two-photon absorption, making this technique attractive for some experiments. Three-photon excitation can enhance z-axis resolution to an even greater degree than two-photon absorption. This is due to a smaller cross section for fluorophore excitation caused by the requirement for simultaneous interaction with three individual photons. In practice, a laser emitting infrared light with a wavelength distribution centered at 1050 nanometers is able to excite a fluorophore that absorbs in the ultraviolet region (approximately 350 nanometers, one-third of the excitation wavelength). The same laser can simultaneously excite another fluorophore at half the wavelength (525 nanometers), a useful combination in dual-labeled biological experiments.

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