The difference between (two-photon, confocal) fluorescence microscope and ordinary microscope
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.
Knowledge related to confocal fluorescence microscopy
The basic principle of confocal fluorescence microscopy is to use a point light source to irradiate the specimen, forming a well-defined small light spot on the focal plane. The fluorescence emitted from this spot after irradiation is collected by the objective lens and sent back along the original irradiation path to the beam splitter composed of a dichroic mirror. The spectrometer sends fluorescence directly to the detector. There is a pinhole in front of both the light source and the detector, called the illumination pinhole and the detection pinhole, respectively. The geometric dimensions of the two are consistent, approximately 100-200nm; Compared to the light spot on the focal plane, the two are conjugate, meaning that the light spot passes through a series of lenses and can ultimately focus on both the illumination pinhole and the detection pinhole simultaneously. In this way, light from the focal plane can converge within the range of the detection hole, while scattered light from above or below the focal plane is blocked outside the detection hole and cannot be imaged. Scanning the sample point by point with a laser, the photomultiplier tube after detecting the pinhole also obtains the corresponding confocal image of the light spot point by point, converts it into a digital signal and transmits it to the computer, and finally aggregates it into a clear confocal image of the entire focal plane on the screen.
