Development Trend of Confocal Microscopy
In order to obtain better observation results at the technical level, confocal microscopy focuses on improving the technical level from the aspects of improving resolution, reducing phototoxicity, increasing scanning speed, observing thick samples, and observing in vivo. At present, it is Z effective. What promotes Zda for confocal microscopy technology is ultra-high resolution microscopy, which breaks through the optical limit and allows researchers to obtain finer cell structures, which is conducive to researchers' deeper understanding of life activities. The next research focus of confocal microscopy technology should focus on the following points:
1. Faster scanning speed
At present, the confocal scanning speed is limited by the mechanical structure of the scanning equipment, and the resolution can only be sacrificed in order to obtain a faster scanning speed. Many biological processes are so rapid that they remain undetectable.
2. More perfect ultra-high resolution technology
At present, ultra-high resolution technologies include STORM, PALM, STED, and SSIM, with resolutions ranging from 20 to 200 nm, but each technology has certain defects, such as sample processing, Z-axis direction, phototoxicity, etc. It is not nearly perfect, and it is often difficult to achieve the limit resolution in actual observation.
3. Stronger compatibility
Confocal microscopy technology involves a variety of excitation methods, such as the multiphoton and light sheet observation mentioned above, and coherent anti-Stokes Raman scattering can theoretically share a set of laser systems. Super-resolution microscopy can be paired with white laser technology. However, due to the patent issues of various companies, there is still room for improvement in terms of completeness and compatibility, and the current equipment cannot give full play to the technical advantages of the application.
Principles of Confocal Microscopy
Confocal microscope is composed of four parts: microscope optical system, laser light source, scanner and detection and processing system. It uses laser with good coherence as light source. It adopts conjugate focusing principle and device on the basis of traditional optical microscope, and uses computer A set of observation, analysis and output system for image processing.
The laser scanning beam passes through the pinhole of the grating to form a point light source, which is reflected to the objective lens through the beam splitter, focused on the specimen and scanned. After the sample is excited, the emitted fluorescence returns to the spectroscope and gathers it to the detection pinhole, and then it is converted into an electrical signal by the photomultiplier tube and transmitted to the computer to display a clear focal plane image. The excitation light is focused on the sample through the pinhole of the grating, and the fluorescence is focused on the pinhole through the objective lens. This process forms two focusing, so it is called a confocal microscope.
Ordinary biological samples have a complex structure and a certain thickness. When observed with an ordinary fluorescence microscope, the fluorescence emitted by the sample overlaps with each other, and the image resolution is greatly reduced.
The confocal imaging of the confocal microscope can effectively suppress the stray light and non-measurement light outside the focal plane from entering the detector, realize single focal plane imaging, and greatly improve the resolution. When the stage moves uniformly in the horizontal direction, it can form a clear single-layer image, and in the vertical direction, it can realize the layer-by-layer scanning of the sample at different depths, and obtain the three-dimensional structure of the sample after three-dimensional reconstruction, which is the so-called "optical CT". The sample is labeled with a fluorescent probe and then observed with a confocal microscope. Not only can various fixed cells and tissue structures be observed, but also the morphology, structure, and ions of living cells can be qualitatively and quantitatively observed and measured regularly.
