What is Two-Photon Fluorescence Microscopy
The basic principle of two-photon excitation is: in the case of high photon density, fluorescent molecules can absorb two long-wavelength photons at the same time, 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 whose wavelength is half the long wavelength to excite a fluorescent molecule. Two-photon excitation requires a high photon density. In order not to damage cells, two-photon microscopy uses high-energy mode-locked pulsed lasers. The laser emitted by this laser has high peak energy and low average energy, its pulse width is only 100 femtoseconds, and its frequency can reach 80 to 100 megahertz. When using a high numerical aperture objective lens to focus the photons of the pulsed laser, the photon density at the focal point of the objective lens is the highest, and the two-photon excitation only occurs at the focal point of the objective lens, so the two-photon microscope does not need a confocal pinhole, which improves the Fluorescence detection efficiency.
In general fluorescence phenomena, due to the low photon density of the excitation light, a fluorescent molecule can only absorb one photon at the same time, and then emit a fluorescent photon through a radiative transition, which is single-photon fluorescence. For the fluorescence excitation process using laser as the light source, two-photon or even multi-photon fluorescence may occur. At this time, the intensity of the excitation light source used is high, and the photon density meets the requirements of fluorescent molecules absorbing two photons at the same time. In the process of using a general laser as the excitation light source, the photon density is still not enough to produce the two-photon absorption phenomenon. Usually, a femtosecond pulsed laser is used, and its instantaneous power can reach the order of megawatts. Therefore, the wavelength of two-photon fluorescence is shorter than the wavelength of excitation light, which is equivalent to the effect produced by excitation at half the excitation wavelength.
