A traditional light microscope consists of several parts
Traditional optical microscopes are mainly composed of optical systems and their supporting mechanical structures. The optical systems include objective lenses, eyepieces and condenser lenses, all of which are complicated magnifying glasses made of various optical glasses. The objective lens enlarges the image of the specimen, and its magnification M object is determined by the following formula: M object = Δ∕f' object , where f' object is the focal length of the objective lens, and Δ can be understood as the distance between the objective lens and the eyepiece. The eyepiece magnifies the image formed by the objective lens again, and forms a virtual image at 250mm in front of the human eye for observation. This is the most comfortable observation position for most people. The magnification of the eyepiece M eye=250/f' eye, f' eye is the eyepiece focal length. The total magnification of the microscope is the product of the objective lens and the eyepiece, that is, M=M object*M eye=Δ*250/f' eye *f; object. It can be seen that reducing the focal length of the objective lens and the eyepiece will increase the total magnification, which is the key to seeing bacteria and other microorganisms with a microscope, and it is also the difference between it and ordinary magnifying glasses.
So, is it conceivable to reduce the f' object f' mesh without limit, so as to increase the magnification, so that we can see more subtle objects? The answer is no! This is because the light used for imaging is essentially a kind of electromagnetic wave, so diffraction and interference phenomena will inevitably occur during the propagation process, just like the ripples on the water surface that can be seen in daily life can go around when encountering obstacles, and two columns of water waves can strengthen each other when they meet Or weaken the same. When the light wave emitted from a point-shaped luminous object enters the objective lens, the frame of the objective lens hinders the propagation of light, resulting in diffraction and interference. There is a series of light rings with weak and gradually weakening intensity. We call the central bright spot as the Airy disk. When two light-emitting points are close to a certain distance, the two light spots will overlap until they cannot be confirmed as two light spots. Rayleigh proposed a judgment standard, thinking that when the distance between the centers of the two light spots is equal to the radius of the Airy disk, the two light spots can be distinguished. After calculation, the distance between the two light-emitting points at this time is e=0.61 入/n.sinA=0.61 I/N.A, where I is the wavelength of light, the wavelength of light that can be received by the human eye is about 0.4-0.7um, and n is the refractive index of the medium where the light-emitting point is located, such as in air, n≈1, in water , n≈1.33, and A is half of the opening angle of the light-emitting point to the frame of the objective lens, and N.A is called the numerical aperture of the objective lens. It can be seen from the above formula that the distance between two points that can be distinguished by the objective lens is limited by the wavelength of light and the numerical aperture. Since the wavelength of the most acute vision of the human eye is about 0.5um, and the angle A cannot exceed 90 degrees, sinA is always less than 1. The maximum refractive index of the available light-transmitting medium is about 1.5, so the e value is always greater than 0.2um, which is the minimum limit distance that the optical microscope can distinguish. Magnify the image through a microscope, if you want to magnify the object point distance e that can be resolved by the objective lens with a certain N.A value enough to be resolved by the human eye, you need M.e ≥ 0.15mm, where 0.15mm is the experimental value of the human eye The minimum distance between two micro-objects that can be distinguished at 250mm in front of the eyes, so M≥ (0.15∕0.61 in) N.A≈500N.A, in order to make the observation not too laborious, it is enough to double the M, that is, 500N. A≤M≤1000N.A is a reasonable selection range of the total magnification of the microscope. No matter how large the total magnification is, it is meaningless, because the numerical aperture of the objective lens has limited the minimum resolvable distance, and it is impossible to distinguish more by increasing the magnification. Small objects are detailed.
Imaging contrast is another key issue of optical microscopes. The so-called contrast refers to the black-and-white contrast or color difference between adjacent parts on the image surface. It is difficult for the human eye to judge the brightness difference below 0.02. is slightly more sensitive. For some microscope observation objects, such as biological specimens, the brightness difference between the details is very small, and the design and manufacturing errors of the microscope optical system further reduce the imaging contrast and make it difficult to distinguish. At this time, the details of the object cannot be seen clearly, not because the total magnification is too low , nor is the numerical aperture of the objective lens too small, but because the contrast of the image plane is too low.
Over the years, people have worked hard to improve the resolution and imaging contrast of the microscope. With the continuous advancement of computer technology and tools, the theory and methods of optical design are also continuously improved. Coupled with the improvement of raw material performance, process and The continuous improvement of detection methods and the innovation of observation methods have made the imaging quality of the optical microscope close to the perfection of the diffraction limit. People will use specimen staining, dark field, phase contrast, fluorescence, interference, polarization and other observation techniques to make the optical microscope It can adapt to the research of all kinds of specimens. Although electron microscopes, ultrasonic microscopes and other magnifying imaging instruments have come out successively in recent years, and have superior performance in some aspects, they are still not available in terms of cheapness, convenience, intuition, and especially suitable for research on living organisms. Rival to the light microscope, which still holds its ground firmly. On the other hand, combined with laser, computer, new material technology and information technology, the ancient optical microscope is rejuvenating and showing vigorous vitality. Digital microscope, laser confocal scanning microscope, near-field scanning microscope, two-photon microscope and There are various new functions or instruments that can adapt to various new environmental conditions emerge in an endless stream, which further expands the application field of optical microscopes. How exciting are the microscopic pictures of rock formations uploaded from the Mars rovers! We can fully believe that the optical microscope will benefit mankind with an updated attitude.
