Benefits and Limitations of Multiphoton Laser-Scanning Microscopy
Advantages
① Excitation is performed with red or infrared light, leading to low light scattering. Scattering by small particles is inversely proportional to the fourth power of the wavelength.
② No pinhole is required, enabling more scattered photons from the imaging plane to be collected.
③ A pinhole cannot distinguish scattered photons emitted from out-of-focus regions or the focal point. Multiphoton microscopy achieves an excellent signal-to-noise ratio for deep imaging.
④ Ultraviolet or visible light used for 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 moisture, ion concentration, and the 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 have many other merits. In terms of 3D resolution, deep penetration, scattering efficiency, background light, signal-to-noise ratio and controllability, they possess superior characteristics unavailable or incomparable to conventional laser microscopes.
Multiphoton confocal laser-scanning microscopes have been extended to various research and application fields. They enable three-dimensional non-invasive observation of samples under natural conditions and improve the resolution and signal-to-noise ratio of the system. By utilizing changes in material properties induced by multiphoton excitation, 3D high-density data storage and microfabrication in arbitrary three-dimensional directions can also be realized, presenting high application value. It is expected that with further advances in mechanics, materials, laser technology and other fields related to multiphoton confocal microscopy, multiphoton confocal laser-scanning microscopes will achieve greater development and wider application.
Limitations
① It only supports fluorescence imaging.
② If the sample contains chromophores that absorb excitation light (such as pigments), thermal damage to the sample may occur.
③ The resolution is slightly reduced. Although this can be improved by simultaneously adopting a confocal pinhole, signal loss will occur.
④ Restricted by costly ultrafast lasers, multiphoton scanning microscopes come with a relatively high cost.
