Improved advantages of laser scanning multiphoton microscope
Laser scanning multiphoton microscope is an important improvement of optical microscope, which mainly shows that it can observe the deep structure of living cells, fixed cells and tissues, and can obtain clear and sharp multi-layer Z-plane structure, that is, optical slices, which can be used to construct the three-dimensional solid structure of specimens. Confocal microscope uses laser light source, which fills the whole back focal plane of the objective lens after expansion, and then passes through the lens system of the objective lens to converge into very small points on the focal plane of the specimen. According to the numerical aperture of the objective lens, the diameter of the brightest illumination point is about 0.25 ~ 0.8μm and the depth is about 0.5 ~ 1.5 μ m.. The size of copolymerization focus depends on microscope design, laser wavelength, objective lens characteristics, scanning unit state setting and specimen properties. The illumination range and depth of field microscope are very large, while the illumination of confocal microscope is concentrated on a * * focus on the focal plane. The most basic advantage of confocal microscope is that it can make fine optical slices of thick fluorescent specimens (which can reach 50 μm or more), and the thickness of the slices is about 0.5 to 1.5 μ m. A series of optical slice images can be obtained by moving the specimen up and down by the Z-axis stepping motor of the microscope. The acquisition of image information is controlled in the first plane and will not be disturbed by signals from other positions on the specimen. After removing the influence of background fluorescence and increasing the signal-to-noise ratio, the contrast and resolution of confocal image are obviously improved compared with traditional field illumination fluorescence image. In many specimens, many intricate structural components are intertwined to form a complex system, but once enough optical sections can be collected, we can reconstruct them in three dimensions by software. This experimental method has been widely used in biological research to clarify the complex structural and functional relationship between cells or tissues.
