Improved Advantages of Laser Scanning Multiphoton Microscopy
Laser scanning multiphoton microscopy is a major improvement of optical microscopy, mainly for observing the deep structure of living cells, fixed cells and tissues, and can obtain clear and sharp multi-layer Z-plane structures, that is, optical slices, and can construct The three-dimensional solid structure of the specimen. The confocal microscope uses a laser light source, which is expanded to fill the entire rear focal plane of the objective lens, and then converges into a very small point on the focal plane of the specimen through the lens system of the objective lens. Depending on the numerical aperture of the objective lens, the diameter of the brightest illumination point is about 0.25 ~ 0.8 μm, and the depth is about 0.5 ~ 1.5 μm. The size of the confocal spot depends on the design of the microscope, the laser wavelength, the characteristics of the objective lens, the state setting of the scanning unit and the nature of the specimen. Field microscopes have a large range of illumination and a large depth of illumination, while confocal microscopes have illumination that is concentrated at a focal point on the focal plane. The most basic advantage of confocal microscopy is that it can perform fine optical sectioning of thick fluorescent specimens (up to 50 μm or more), with a thickness of about 0.5 to 1.5 μm. A series of optical section images can be obtained by moving the specimen up and down with the microscope Z-axis stepper motor. The acquisition of image information is controlled within the plane and will not be interfered by signals from other locations on the specimen. After removing the effect of background fluorescence and increasing the signal-to-noise ratio, the contrast and resolution of confocal images are significantly improved compared with traditional field illumination fluorescence images. In many specimens, many intricate structural components are intertwined to form complex systems, but once enough optical sections can be collected, we can reconstruct them in 3D through software. This experimental approach has been widely used in biological research to elucidate the complex structural and functional relationships between cells or tissues.
