Advantages and Disadvantages of Multiphoton Laser Scanning Microscopy (MLSM)
Multiphoton laser scanning microscopy is an experimental method based on laser scanning microscopy technology, which provides better optical sectioning capabilities for three-dimensional observation. The multi-photon fluorescence excitation method uses long-wavelength red light or near-infrared light to collect high-resolution fluorescence images of specimens, which causes minimal damage to living specimens. Therefore, it is suitable for live cell imaging, especially thick living tissues such as brain slices, embryos, etc. Imaging studies of whole organs or even entire bodies.
The advantages are as follows:
1. Use red light or infrared light to excite, and the light scattering is small (the scattering of small particles is inversely proportional to the fourth power of the wavelength).
2. No pinhole is needed, and more scattered photons from the imaging cross-section can be collected.
3. The pinhole cannot distinguish scattered photons emitted from the defocused area or the focus area, and the signal-to-noise ratio of multi-photon imaging in deep layers is good.
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, and is difficult to excite deep layers.
5. In terms of biological microscope observation, the first consideration is not to damage the active state of the organism itself and to maintain the flow of water, ion concentration, oxygen and nutrients. In the case of light observation, both heat and photon energy must stay within the irradiation amount and light energy that the cells will not be damaged.
6. Multiphoton microscopy also has many advantages. For example, in terms of three-dimensional resolution, depth penetration, scattering efficiency, background light, signal-to-noise ratio, control, etc., there are transcendent characteristics that previous laser microscopes did not have or could not match.
Multi-photon confocal laser scanning microscopy has been extended to various research and application fields. It can conduct three-dimensional non-destructive observation of samples in their natural state, and can improve the resolution and signal-to-noise ratio of the system. Using multi-photon excitation materials Changes in properties can also realize three-dimensional height data storage and three-dimensional micro-machining in any direction, which has high application value. It is believed that with the further development of machinery, materials, laser technology, etc. related to multi-photon confocal microscopy, more Photonic confocal laser scanning microscopy will receive greater development and wider application.
The disadvantages are as follows:
1. It can only image fluorescence.
2. If the sample contains chromophores that can absorb excitation light, such as pigments, the sample may be damaged by heat.
3. The resolution is slightly reduced. Although it can be improved by using confocal holes at the same time, there will be signal loss.
4. Limited by expensive ultrafast lasers, the cost of multiphoton scanning microscopes is relatively high.
