Introduction to the Advantages and Limitations of Multiphoton Laser Scanning Microscopy
Multiphoton laser scanning microscopy is an experimental technique developed on the basis of laser scanning microscopy. It delivers superior optical sectioning performance for three-dimensional observation.
The multiphoton fluorescence excitation method employs long-wavelength red light or near-infrared light to acquire high-resolution fluorescence images of specimens. It causes minimal damage to living samples and is therefore suitable for live cell imaging, especially for the imaging research of thick living tissues such as brain slices, embryos, entire organs and even complete organisms.
Its main advantages are summarized as follows:
Excitation is performed with red or infrared light, which features low light scattering. (Scattering by small particles is inversely proportional to the fourth power of the wavelength.)
No pinhole is required, enabling more efficient collection of scattered photons from the imaging plane.
A pinhole cannot distinguish scattered photons emitted from defocused areas versus the focal zone; multiphoton microscopy provides an excellent signal-to-noise ratio for deep-tissue imaging.
Ultraviolet or visible light used in single-photon excitation is easily absorbed and attenuated by samples before reaching the focal plane, making deep excitation difficult to achieve.
In biological microscopic observation, the primary consideration is to preserve the physiological activity of organisms, maintaining normal water content, ion concentration, and the circulation of oxygen and nutrients. During optical observation, both heat generation and photon energy must be kept within exposure levels that do not cause cellular damage.
Multiphoton microscopy possesses numerous additional strengths. It exhibits unparalleled advantages over conventional laser microscopes in terms of three-dimensional resolution, deep penetration depth, scattering efficiency, background noise suppression, signal-to-noise ratio and imaging controllability.
Multiphoton confocal laser scanning microscopy has been applied in a wide range of research and practical fields. It allows non-invasive three-dimensional observation of samples under natural conditions while improving the resolution and signal-to-noise ratio of the system. By utilizing material property changes induced by multiphoton excitation, it also enables high-density three-dimensional data storage and microfabrication in arbitrary three-dimensional directions, demonstrating great application value.
It is foreseeable that with the further advancement of mechanics, material science, laser technology and other related disciplines, multiphoton confocal laser scanning microscopy will achieve greater progress and wider application prospects.
