Introduction to the advantages and disadvantages of multiphoton laser scanning microscopy
1. Using red light or infrared light to excite, the light scattering is small, and the scattering of small particles is inversely proportional to the fourth power of the wavelength.
2. More scattered photons from the imaging cross-section can be collected without the need for pinholes.
3. The pinhole cannot distinguish scattered photons emitted from the defocused 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 is difficult 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 flow 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 the cells will not be damaged.
6. Multiphoton microscopy has many advantages. Such as three-dimensional resolution, depth penetration, scattering efficiency, background light, signal-to-noise ratio, control, etc., all have characteristics that previous laser microscopes did not have or could not compare with.
Disadvantages of multiphoton laser scanning microscopy:
1. It can only image fluorescence.
2. If the sample contains chromophores that absorb excitation light, the sample may be thermally damaged.
3. The resolution is slightly reduced. Although it can be improved by using confocal holes at the same time, there is signal loss.
4. Limited by expensive ultrafast lasers, the cost of multiphoton scanning microscopes is relatively high.
