Introduction to the principle and performance of ordinary optical microscope

(1) Imaging principle of optical microscope
The magnification of the microscope is done by a lens, and the single lens imaging has aberrations that affect the image quality. A lens group composed of a single lens is equivalent to a convex lens, and the amplification effect is better.

(two) the performance of the microscope
The resolution of the microscope depends on various conditions of the optical system. The object to be observed must have a high magnification and be clear. After the object is enlarged, whether it can present a clear fine structure depends first on the performance of the objective lens, and secondly on the performance of the eyepiece and the concentrating mirror.

1. The numerical aperture is also called the aperture ratio (or aperture ratio), abbreviated as NA. Both the objective lens and the concentrator are marked with their numerical aperture. The numerical aperture is the main parameter of the objective lens and the concentrator, and is also to judge their performance. The most important indicator. The numerical aperture is closely related to the various properties of the microscope. It is proportional to the resolution of the microscope, inversely proportional to the depth of focus, and proportional to the square root of the brightness of the image.
The numerical aperture can be expressed by the following formula:

In the formula:
N—mediation rate between the objective lens and the specimen
——the mirror angle of the objective lens

The so-called mirror angle refers to the angle between the light emitted from the object point on the optical axis of the objective lens and the edge of the effective diameter of the front lens of the objective lens, as shown in Figure 1-5.
The mirror angle α is always less than 180°. Since the refractive index of air is 1, the numerical aperture of the dry objective lens is always less than 1, typically 0.05-0.95; if the oil immersion objective is immersed with cedar oil (refractive index 1.515), the numerical aperture can be as close as 1.5. Although the theoretical numerical aperture limit is equal to the refractive index of the immersion medium used, it is actually impossible to achieve this limit from the manufacturing technique of the lens. The maximum numerical aperture of a premium oil immersion objective is typically 1.4 in practical applications.

The refractive indices of the media of several substances are as follows:
The air was 1.0, the water was 1.33, the glass was 1.5, the glycerin was 1.47, and the cedar was 1.52.
The effect of the refractive index of the medium on the light path of the objective lens is shown in Figure 1-6.

2, resolution
D can be expressed by the following formula:
D=λ/2N.A.
The wavelength of visible light is 0.4-0.7 microns and the average wavelength is 0.55 microns. If an objective lens having a numerical aperture of 0.65 is used, then D = 0.55 microns / 2 x 0.65 = 0.42 microns. This means that the object to be inspected can be observed at 0.42 μm or more, and if it is less than 0.42 μm, it cannot be seen. If an objective lens with a numerical aperture of 1.25 is used, then D = 2.20 microns. Where the length of the object to be inspected is greater than this value, it can be seen. It can be seen that the smaller the D value, the higher the resolution and the clearer the image. According to the above formula, it is possible to: (1) reduce the wavelength; (2) increase the refractive index; (3) increase the mirror angle to improve the resolution. Microscopes and electron microscopes that use ultraviolet light as a light source use short light waves to improve resolution to view smaller objects. The resolution of the objective lens is closely related to whether the image is clear or not. The eyepiece does not have this property. The eyepiece only magnifies the image created by the objective lens.

3. Magnification:
The microscope magnifies the object, first through the objective lens for the first time to enlarge the image, and the eyepiece causes a second magnification image at the bright distance. Magnification is the ratio of the size of the final image to the original object. Therefore, the magnification (V) of the microscope is equal to the product of the objective magnification (V1) and the eyepiece magnification (V2), namely:
V=V1×V2
A more accurate calculation method can be obtained from the following formula

F1=the focal length of the objective lens, F2=the focal length of the eyepiece â–³=the length of the optical tube, D=the distance of the eyesight (=250 mm)

Let â–³=160 mm F1=4 mm D=250 mm F2=150 mm

4, depth of focus:
When observing a specimen under a microscope, when the focus is on a certain image surface, the image is the clearest, and the image surface is the target surface. In addition to the target surface in the field of view, blurred objects can be seen above and below the target surface, and the distance between the two surfaces is called the depth of focus. The focal depth of the objective lens is inversely proportional to the numerical aperture and the magnification: that is, the larger the numerical aperture and the magnification, the smaller the focal depth. Therefore, adjusting the oil mirror is more careful than adjusting the low power mirror, otherwise it is easy to make the object slide over and cannot be found.

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