Introduction to the Advantages and Disadvantages of Multiphoton Laser Scanning Microscope
Advantages of Multiphoton Laser Scanning Microscopy:
1. Excited by red light or infrared light, 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 section can be collected without pinholes.
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 has many advantages. Such as three-dimensional resolution, deep penetration, scattering efficiency, background light, signal-to-noise ratio, control, etc., all have characteristics that previous laser microscopes did not have or could not surpass.
Disadvantages of Multiphoton Laser Scanning Microscopy:
1. Only for fluorescence imaging.
2. If the sample includes chromophores that absorb excitation light, 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 is lost.
4. Due to the limitation of expensive ultrafast lasers, the cost of multiphoton scanning microscope is relatively high.
