Knowledge about Two-Photon Fluorescence Microscopy
The basic principle of two-photon excitation is that at high photon density, fluorescent molecules can simultaneously absorb two long wavelength photons and emit a shorter wavelength photon after a short period of so-called excited state lifetime; The effect is the same as using a photon with half the wavelength of the long wavelength to excite fluorescent molecules. Two photon excitation requires a high photon density, and in order to avoid damaging cells, two-photon microscopy uses high-energy mode-locked pulse lasers. The laser emitted by this laser has high peak energy and low average energy, with a pulse width of only 100 femtoseconds and a frequency of up to 80 to 100 megahertz. When using a high numerical aperture objective lens to focus the photons of a pulsed laser, the photon density at the focal point of the objective lens is the highest, and two-photon excitation only occurs at the focal point of the objective lens. Therefore, the two-photon microscope does not require a confocal pinhole, which improves the efficiency of fluorescence detection.
In general fluorescence phenomena, due to the low photon density of the excitation light, a fluorescent molecule can only absorb one photon at a time and then emit another fluorescent photon through radiative transition, which is known as single photon fluorescence. For fluorescence excitation processes using lasers as light sources, two-photon or even multiphoton fluorescence phenomena may occur. In this case, the excitation light source used has high intensity and photon density that meets the requirement for fluorescent molecules to absorb two photons simultaneously. In the process of using a general laser as the excitation light source, the photon density is still insufficient to produce two-photon absorption phenomenon. Typically, femtosecond pulse lasers are used, with instantaneous power reaching the megawatt level. Therefore, the wavelength of two-photon fluorescence is shorter than that of excitation light, equivalent to the effect produced by half excitation wavelength excitation.
