In the late 1980s, as the advanced However, laser confocal microscopy has a major defect: the confocal aperture not only blocks the fluorescence generated outside the focal point, but also blocks the fluorescence generated by the focal point that is scattered by the biological tissues, resulting in a decrease in the collection efficiency of fluorescence, and the imaging depth can not be more than 100 micrometers. Therefore, in the 1990s, two-photon fluorescence microscopy combining laser confocal microscopy and two-photon excitation technology came into being. The excitation wavelength of the two-photon absorption process is generally set in the bio-optical window range of 680-1080 nm, which avoids the damage of ultraviolet light to the cells or living organisms and penetrates deeper.
Currently, the most widely used two-photon fluorescence microscope is the femtosecond titanium gemstone laser, which is bulky and expensive, which limits the application of two-photon fluorescence microscopy in various fields such as life sciences, chemistry and medicine. So in some fields such as medical diagnostics, people have tried to use compact and affordable sub-nanosecond solid-state lasers as the standard light source.
The basic principle of two-photon excitation is that, when a fluorescent molecule is excited under high photon density, it simultaneously absorbs two long-wavelength photons, and then spontaneously radiates fluorescent photons back to the ground state after excitation leaps and relaxation processes. Compared with conventional single-photon excitation fluorescence microscopy, the optical signal generation in two-photon excitation fluorescence microscopy is nonlinear, with the excitation light being a longer-wavelength, higher-peak-power light source, and the emitted fluorescent photons having a wavelength slightly longer than half of the excitation wavelength.
Two-Photon Fluorescence Microscopy uses a near-infrared laser light source, and its nonlinear nature does not require a confocal aperture. Therefore, compared with confocal microscopy, two-photon fluorescence microscopy has the following advantages:
- Large imaging depth and low optical damage;
- No confocal aperture is required, and the fluorescence collection efficiency is greatly improved;
- Higher spatial resolution and contrast;
- Small excitation range, small phototoxicity and bleaching;
- The emission and excitation wavelengths are far apart, avoiding spectral overlap;
