Technical characteristics and usage skills of two-photon fluorescence microscope
Two-photon fluorescence microscopy is a new technology that combines laser scanning confocal microscopy and two-photon excitation technology.
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 with a wavelength 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 period 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. It is an important research method in the fields of morphology, molecular cell biology, neuroscience, and pharmacology.
1. The background of the emergence of two-photon microscopy - two limitations of traditional laser confocal microscopy:
1) One is the phototoxicity phenomenon: because the confocal pinhole must be small enough to obtain a high-resolution image, and the small aperture will block a large part of the fluorescence emitted from the sample, including the fluorescence emitted from the focal plane, the corresponding Yes, the excitation light must be strong enough to obtain a sufficient signal-to-noise ratio; and the high-intensity laser will cause the fluorescent dye to fade rapidly during continuous scanning, and the fluorescent signal will become weaker and weaker as the scanning progresses.
2) Phototoxicity is another problem. Under laser irradiation, many fluorescent dye molecules will produce cytotoxins such as singlet oxygen or free radicals, so the scanning time and the optical power density of the excitation light should be limited in the experiment to maintain the sample density. active. In the research on active samples, especially the various stages of the growth and development of active samples, photobleaching and phototoxicity make these studies very limited.
2. Why do you say that two-photon microscopes generally do not need to be equipped with ultraviolet excitation lasers?
Two-photon microscopy is a fluorescence excitation technology based on the two-photon excitation effect: fluorescent dye molecules can be excited by absorbing two low-energy photons at the same time (the time interval between two photons reaching fluorescent molecules is less than 1 femtosecond ), its excitation effect can be equivalent to absorbing a high-energy photon of 1/2 wavelength. For example, absorbing two photons at red wavelengths is equivalent to a molecule absorbing ultraviolet. Long-wavelength photons are not easily absorbed by cells, so phototoxicity to living cells is reduced, and photobleaching is also reduced. In this way, it not only plays the function of ultraviolet excitation, but also avoids the damage of ultraviolet light to the sample.
3. What is special about the laser of two-photon microscope?
The probability of two-photon absorption depends on how closely the two incident photons coincide in space and time (the two photons must arrive within 10-18 seconds). The two-photon absorption cross section is small, and only fluorophores in regions with a large photon flux are excited. Therefore, most of the lasers used are titanium sapphire lasers, which can achieve picosecond or femtosecond scanning speeds, and have very high peak power and low average power, so that photobleaching and phototoxicity can be reduced or eliminated. The most important thing is to provide a very high density of photons in a small range, which can ensure the simultaneous excitation of two photons.
4. What are the advantages of two-photon excitation?
1) Increase the selectivity of the dye: the excitation light range of the confocal system laser (Ar, Ar/Kr, HeNe) is 488nm - 647nm. This means experimenting with UV-excited fluorescent dyes, eg with DAPI , Hoescht. The excitation wavelength of two-photon is twice that of single-photon, so dyes excited by ultraviolet can be excited by near-infrared light.
2) Reduce photobleaching: Because of the reduction in photobleaching, the success rate of experiments using CFP/YFP for fluorescence resonance energy transfer (FRET) increases.
3) No special objective lens is required: From the hardware point of view, the excitation of UV-excited dyes with the wavelength of near-infrared light does not require special UV optical components.
4) Improve the signal-to-noise ratio: the excitation light wavelength and the emitted light wavelength have a large difference, which improves the signal-to-noise ratio.
5) Bleaching localized to the focal point: Since fluorescence excitation occurs only at the focal point of the objective, there is no need for a confocal pinhole. This improves light detection and photobleaching occurs only at the focal point.
6) Easier to penetrate specimens: Infrared wavelength light is not easily scattered by cells and can penetrate deeper specimens.
5. Compared with laser scanning confocal microscopy, what is the biggest improvement made by two-photon microscopy?
1) Reduced photobleaching.
2) Reduced phototoxicity.
3) It is not easy to scatter, and it is easier to penetrate thick samples, such as brain slices.
