Enhancing the benefits of laser scanning multiphoton microscopy
Laser scanning multiphoton microscopy is a major improvement over optical microscopy. It can observe the deep structure of living cells, fixed cells and tissues, and can obtain clear and sharp multi-layer Z-plane structures, that is, optical sections, from which it can construct The three-dimensional solid structure of the specimen. Confocal microscopy uses a laser light source that, after expansion, fills the entire back focal plane of the objective lens, and then passes through the lens system of the objective lens to converge into a very small point on the focal plane of the specimen. Depending on 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 ~ 1.5μm. The size of the confocal spot depends on the microscope design, laser wavelength, objective lens characteristics, scanning unit status settings and specimen properties. Field microscopy has a large illumination range and depth, whereas confocal microscopy has a focused illumination focused on 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 (which can reach 50 μm or more), and the thickness of the sections is about 0.5 to 1.5 μm. A series of optical section images can be obtained by moving the specimen up and down using the microscope's Z-axis stepper motor. The acquisition of image information is controlled within the plane and will not be interfered by signals emitted from other locations on the specimen. After removing the influence of background fluorescence and increasing the signal-to-noise ratio, the contrast and resolution of confocal images are significantly improved compared to traditional field-illuminated 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 three dimensions through software. This experimental method has been widely used in biological research to elucidate the complex structural and functional relationships between cells or tissues.
