Advantages and Limitations of Multiphoton Laser Scanning Microscopes
① Excitation is performed with red or infrared light, resulting in low light scattering. Scattering by small particles is inversely proportional to the fourth power of the wavelength.
② Without a pinhole, more scattered photons from the imaging plane can be collected.
③ Pinholes cannot distinguish scattered photons emitted from out-of-focus regions or the focal point. Multiphoton microscopy delivers good signal-to-noise ratio for deep-tissue imaging.
④ Ultraviolet or visible light used in single-photon excitation tends to be absorbed and attenuated by the sample before the beam reaches the focal plane, making deep-layer excitation difficult.
⑤ For biological microscopic observation, the primary consideration is preserving the biological activity of specimens, maintaining water content, ion concentration, and circulation of oxygen and nutrients. For optical observation, both heat and photon energy must be kept within irradiation levels that do not damage cells.
⑥ Multiphoton microscopes possess numerous advantages. In terms of 3D resolution, deep penetration, scattering efficiency, background light, signal-to-noise ratio and controllability, they exhibit superior properties that conventional laser microscopes cannot match or achieve.
Disadvantages of Multiphoton Laser Scanning Microscopes
① It can only perform fluorescence imaging.
② If the sample contains chromophores that absorb excitation light, thermal damage to the sample may occur.
③ The resolution is slightly reduced. Although this can be improved by simultaneously using a confocal pinhole, signal loss will take place.
④ Restricted by expensive ultrafast lasers, multiphoton scanning microscopes have a relatively high cost.
