Wprowadzenie do zalet i wad wielofotonowej mikroskopii skaningowej laserowej
Advantages of multiphoton laser scanning microscope:
1. Excited by red or infrared light, 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. Pinholes cannot distinguish scattered photons emitted from defocused or focal areas, and multiphotons have better signal-to-noise ratio in deep imaging.
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, making it difficult to excite deep layers.
5. In terms of biological microscopy observation, the first consideration is to not damage the active state of the organism itself, maintain the circulation of water, ion concentration, oxygen, and nutrients. In the field of light observation, both thermal and photon energy must remain within the irradiation dose and light energy that do not damage cells.
6. Multiphoton microscopy has many advantages. In terms of three-dimensional resolution, depth invasion, scattering efficiency, background light, signal-to-noise ratio, control, etc., there are characteristics that previous laser microscopes do not possess or cannot surpass
Disadvantages of multiphoton laser scanning microscope:
1. Only for fluorescence imaging.
2. If the sample includes chromophores that can absorb excitation light, the sample may be subjected to thermal damage.
3. The resolution is slightly reduced, although it can be improved by simultaneously utilizing confocal small holes, there is signal loss.
4. Due to the limitations of expensive ultrafast lasers, the cost of multiphoton scanning microscopes is relatively high.
