Introduction to some knowledge about optical microscope
An instrument or device that magnifies a tiny object or a tiny part of an object at a high magnification for observation. It is widely used in industrial and agricultural production and scientific research. Biologists and medical workers also use microscopes a lot in their business. Broadly divided into optical microscopes and electron microscopes.
Optical microscope is a microscope that uses visible light as the light source. Ordinary optical microscopes can be divided into two parts: the optical system and the mechanical device. The optical system mainly includes eyepieces, objective lenses, condensers, diaphragms and light sources. The mechanical device mainly includes the lens barrel, mirror column, stage, mirror base, thickness adjustment screw and other parts (Figure 1). Its basic optical principle is shown in Figure 2. The small convex lens on the left in the figure represents a group of lenses with a short focal length, called the objective lens. The large convex lens on the right represents a group of lenses with a long focal length, called the eyepiece. The object to be observed (AB) is placed slightly outside the focal point (f1) of the objective lens. The light from the object forms an inverted magnified real image (B'A') slightly inside the eyepiece focus (f2) after passing through the objective lens. The observer's eyes further magnify the real image (B'A') into an inverted virtual image (B"A") through the eyepiece.
The eyepiece is located above the microscope barrel and generally consists of two convex lenses. In addition to further expanding the real image formed by the objective lens, it also limits the field of view observed by the eyes. According to the magnification, there are three kinds of commonly used eyepieces: 5 times, 10 times and 15 times.
The objective lens is generally located below the microscope barrel, close to the object being observed. It consists of 8 to 10 lenses. Its function is to enlarge (create an enlarged real image for the object), the second is to ensure the quality of the image, and the third is to increase the resolution. Commonly used objective lenses can be divided into low magnification (4×), medium magnification (10× or 20×), high magnification (40×) and oil immersion objective lens (100×) according to the magnification. Multiple objective lenses are mounted on the mirror changer wheel, and the objective lens with different multiples can be selected by rotating the turntable as required.
The magnification of the microscope is the multiple of the eyepiece multiplied by the objective. For example, if the eyepiece is 10 times and the objective lens is 40 times, the magnification is 40×10 times (magnification 400 times). A good microscope can magnify 2000 times and can distinguish two points 1×10-5cm apart.
When white light passes through the convex lens, the light with shorter wavelength (blue-purple) has a greater refraction than the light with long wavelength (red-orange). Therefore, when imaging, there are various spectrums around the image, and there is a circle of blue or red light. This color defect is called chromatic aberration. Due to the different angles at which light enters (and exits) the various parts of the lens surface, the light passing through the periphery of the lens is refracted at a larger angle than the light passing through the center of the lens. Therefore, blurred and distorted images appear around the image circumference during imaging. This defect of imaging surface curvature is called spherical aberration. A series of convex and concave lens groups with different shapes, structures and distances cooperate with each other to correct chromatic aberration and spherical aberration to the greatest extent, forming a bright, clear and accurate image. This is why the eyepiece or the objective lens is composed of a set of lenses respectively. Such lenses are called plan achromats.
When light is projected from one medium (such as air) to another denser medium (such as glass), it will bend to the "normal line" (a line perpendicular to the interface of the medium), such as the BOA line in Figure 3. When light enters from a dense medium (glass) into a non-dense medium (air), it will deviate from the "normal line", such as the AOB line (Figure 3a). When the light passes through the condenser glass (refractive index 1.51) and enters the air, it will also deviate and refract outward, so the amount of light entering the objective lens is greatly reduced, and the resolution of the image is also reduced. When using a 100x objective lens, if oil is filled between the objective lens and the cover glass (the refractive index is also 1.51) to isolate the air, the light can enter the objective lens almost without refraction, which increases the brightness and resolution of the image . Such objectives are called oil immersion objectives (Figure 3b).
The condenser is located under the microscope stage, which can converge the light from the source of light, concentrate the light on the specimen, and make the specimen evenly irradiated with moderate light intensity. The lower end of the condenser is equipped with an aperture stop (diaphragm) to control the thickness of the beam.
The illumination source of ordinary optical microscope is located under the condenser, which is a special strong light bulb with uniform illumination, and is equipped with a variable resistor to change the intensity of the light.
Since the light source light of an ordinary optical microscope transmits from the bottom of the lens body, passes through the condenser lens, the objective lens, and reaches the eyepiece, the sample to be observed must be cut into thin slices with a thickness of about 6 μm that can transmit light in medical and biological research. And to stain to show different tissues and cells and other fine structures. The whole processing process is called the conventional tissue slice technique, including selecting appropriate tissue materials, fixing them with formaldehyde (formalin) solution, dehydrating with alcohol step by step, embedding in paraffin, cutting the tissue into thin slices with a microtome and mounting them on glass slides, and then After staining with hematoxylin-eosin dye, the tissue slides were finally mounted in optical resin glue. Prepared tissue slides can be stored for a long time.
The eyepiece and objective lens of the microscope are installed at both ends of the lens barrel, and their distance is fixed. Place the tissue slide on the stage, and rotate the coarse adjustment screw to bring the stage close to the objective lens. The tissue slice enters the focal plane of the objective lens, and the tissue image in the specimen can be seen in the eyepiece. Then use the fine adjustment screw to make the image in the eyepiece clear to observe. When changing the magnification, the eyepiece or objective lens must be replaced.
