Advantages and Disadvantages of Laser Scanning Microscopy
The advantages are as follows:
1. Excited by red or infrared light, light scattering is small (the scattering of small particles is inversely proportional to the fourth power of the wavelength).
2. No need for pinholes, able to collect more scattered photons from the imaging cross-section.
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, and to 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 also has many advantages. In terms of three-dimensional resolution, depth invasion, scattering efficiency, background light, signal-to-noise ratio, control, etc., laser microscopes have unparalleled or superior characteristics that previous laser microscopes did not possess.
Multiphoton confocal laser scanning microscopy has been extended to various research and application fields, It can perform three-dimensional non-destructive observation on samples in their natural state and improve the resolution and signal-to-noise ratio of the system. By utilizing the changes in material properties after multi photon excitation, it can also achieve three-dimensional height data storage and three-dimensional micro processing in any direction, which has high application value It can be believed that with the further development of mechanical, material, and laser technologies related to multiphoton confocal microscopy, multiphoton confocal laser scanning microscopy will have greater development and wider applications
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 subjected to thermal damage.
3. The resolution is slightly reduced, although it can be improved by simultaneously utilizing confocal small holes, there will be signal loss.
4. Due to the limitations of expensive ultrafast lasers, the cost of multiphoton scanning microscopes is relatively high.
