Advantages and Disadvantages of Multiphoton Laser Scanning Microscopy
Multiphoton laser scanning microscopy is an experimental method based on laser scanning microscopy technology, which provides more accurate optical sectioning capabilities in three-dimensional observation. The multiphoton fluorescence excitation method uses long-wavelength red light or near-infrared light to collect high-resolution fluorescence images of specimens, which has minimal damage to living specimens, so it is suitable for live cell imaging, especially for thick living tissues such as brain slices, embryos, Imaging studies of whole organs or even whole organisms.
The advantages are as follows:
1. Excited by red light or infrared light, the light scattering is small (the scattering of small particles is inversely proportional to the fourth power of the wavelength).
2. There is no need for pinholes, and more scattered photons from the imaging section can be collected.
3. The pinhole cannot distinguish the scattered photons emitted from the out-of-focus area or the focus area, and the signal-to-noise ratio of multi-photon imaging in deep layers is good.
4. The ultraviolet or visible light used for single-photon excitation is easily absorbed and attenuated by the sample before the beam reaches the focal plane, and it is not easy to excite deep layers.
5. In terms of biological microscope observation, the first consideration is not to damage the active state of the organism itself, and to maintain the circulation of water, ion concentration, oxygen and nutrients. In the case of light observation, both heat and photon energy must stay within the irradiation amount and light energy that will not damage the cells.
6. Multiphoton microscopy also has many advantages. Such as three-dimensional resolution, deep penetration, scattering efficiency, background light, signal-to-noise ratio, control, etc., there are superior characteristics that laser microscopes do not have or have incomparable in the past.
Multiphoton confocal laser scanning microscopy has been extended to various research and application fields. It can perform three-dimensional non-destructive observation of samples in the natural state, and can improve the resolution and signal-to-noise ratio of the system. Using multiphoton excitation materials It can also realize three-dimensional height data storage and three-dimensional arbitrary direction microfabrication, which has high application value. It is believed that with the further development of machinery, materials, and laser technology related to multiphoton confocal microscopy, there will be more Photon confocal laser scanning microscope will get greater development and wider application.
The disadvantages are as follows:
1. Only for fluorescence imaging.
2. If the sample includes chromophores that can absorb excitation light, such as pigments, the sample may be thermally damaged.
3. The resolution is slightly reduced. Although it can be improved by using the confocal aperture at the same time, the signal will be lost.
4. Due to the limitation of expensive ultrafast lasers, the cost of multiphoton scanning microscope is relatively high.
