Optical path of ordinary optical microscope
1. An ordinary optical microscope is a precision optical instrument. In the past, simple microscopes consisted of only a few lenses, while today's microscopes consist of a set of lenses. Ordinary optical microscopes can usually magnify objects 1500-2000 times. (1) The structure of the microscope The structure of the ordinary optical microscope can be divided into two parts: one is the mechanical device, and the other is the optical system. Only when these two parts cooperate well can the microscope function. First, the mechanical device of the microscope The mechanical device of the microscope includes the frame, the lens barrel, the objective lens converter, the stage, the push rod, the coarse screw, the micro screw and other components. The bracket consists of a base and a mirror arm. The stage and the lens barrel are attached to it, which is the basis for installing the optical magnification system components.
(2) The eyepiece is connected to the lens barrel of the lens barrel, and the converter is connected to the bottom, forming a dark room between the eyepiece and the objective lens (installed under the converter). The distance from the trailing edge of the objective to the end of the barrel is called the mechanical barrel length. Because the magnification of the objective lens is for a certain length of the lens barrel. Changes in the length of the lens barrel will not only change the magnification, but also affect the image quality. Therefore, when using the microscope, the length of the lens barrel cannot be changed at will. Internationally, the standard barrel length of the microscope is set at 160mm, and this number is marked on the housing of the objective lens.
(3) Objective lens changer The nose lens changer can be equipped with 3 to 4 objective lenses, usually three objective lenses (low magnification, high magnification, oil lens). Nikon microscopes are equipped with four objective lenses. By rotating the converter, any objective lens can be connected to the lens barrel as required, and the eyepiece on the lens barrel constitutes a magnifying system.
(4) There is a hole in the center of the stage, which is the light path. The stage is equipped with spring sample clamps and push rods, whose function is to fix or move the position of the sample so that the microscopic object is just in the center of the field of view.
(5) The pusher is a mechanical device that moves the specimen. It is made of a metal frame with two propulsion gears, one horizontal and one vertical. A good microscope has scales engraved on the bar to create a very precise plane. Coordinate System. If you want to observe a certain part of the test sample repeatedly, you can record the value of the vertical and horizontal ruler during the first inspection, and then move the push rod according to the value to find the position of the original sample.
(6) Coarse spiral is a mechanism that adjusts the distance between the objective lens and the specimen by moving the lens barrel. In old microscopes, after the coarse spiral is twisted forward, the lens descends and approaches the sample. When performing microscopy on new production microscopes, turn the stage forward with the right hand to raise the stage to bring the sample closer to the objective and vice versa.
(7) The micro-movement screw can only use the coarse-movement screw to coarsely adjust the focal length. To get a sharp image, you'll need to make further adjustments with micro-screw. The lens barrel moves 0.1 mm (100 microns) for each revolution of the fretting screw. The thick and thin spirals of the newly produced gao-end microscope are coaxial. 2. The optical system of the microscope The optical system of the microscope consists of a reflector, a condenser, an objective lens, an eyepiece, etc. The optical system magnifies the object to form an enlarged image of the object.
(1) Mirrors Early ordinary optical microscopes used natural light to inspect objects, and a mirror was installed on the frame. The reflector consists of a flat surface and a concave mirror on the other, which can reflect light impinging on it to the center of the condenser lens, thereby illuminating the specimen. When not using a condenser, use a concave mirror. Concave mirrors focus light. When using a condenser, a flat mirror is generally used. The newly produced inferior microscope frame is equipped with a light source and a current adjustment screw, which can adjust the light intensity by adjusting the current.
(2) Condenser The condenser is under the table. It consists of a condenser lens, an iridescent aperture and a lift screw. Concentrators can be divided into brightfield concentrators and darkfield concentrators. Common optical microscopes are equipped with brightfield condensers. Brightfield condensers include Abbe condensers, enlightenment condensers, and falling sand condensers. Abbe condensers suffer from chromatic and spherical aberrations when the objective numerical aperture is higher than 0.6. Qiming condensers are highly corrected for chromatic aberration, spherical aberration and coma. It is a high-quality condenser for brightfield microscopy, but it is not suitable for objectives below 4x. Shaking out the condenser can shake the upper lens of the condenser out of the light path to meet the needs of low magnification objective (4×) large field of view illumination.
The condenser is installed under the stage, and its function is to focus the light reflected by the light source on the sample through the mirror to obtain strong illumination and make the image of the object bright and clear. The height of the condenser is adjustable, so that the focus falls on the object to be inspected, and high brightness is obtained. The focal point of the general condenser is 1.25mm above it, and its rise limit is 0.1mm below the stage plane. Therefore, the thickness of the required glass slide should be between 0.8-1.2mm, otherwise the sample under inspection will not be able to focus, which will affect the microscopic effect. There is also an iridescent aperture in front of the condenser front lens group, which can be opened and closed, affecting the resolution and contrast of the image. If the iris aperture is opened too large, beyond the numerical aperture of the objective, flare will occur; if the aperture is closed down too small, the resolution will be reduced and the contrast will be increased. Therefore, when observing, through the adjustment of the iris aperture, the field diaphragm (microscope with field diaphragm) is opened to the outer tangent of the periphery of the field of view, so that objects not in the field of view cannot get any light. Illumination avoids scattered light interference.
(3) The objective lens installed on the converter at the front end of the lens barrel uses light to make the object under inspection for the first time. The imaging quality of the objective has a decisive influence on the resolution. The performance of an objective depends on the objective's numerical aperture (numerical aperture abbreviated as NA). The numerical aperture of each objective is marked on the housing of the objective. The larger the numerical aperture, the better the performance of the objective. There are many types of objective lenses, which can be classified from different angles: According to the difference in the medium between the front lens of the objective lens and the object to be inspected, it can be divided into: 1. The dry objective lens uses air as the medium, such as the commonly used objective lens below 40×, the numerical aperture is equal to less than 1. ②Oil immersion objectives often use cedar oil as the medium. Such objectives are also called oil lenses. Its magnification is 90×-100×, and the numerical aperture value is greater than 1. According to the magnification of the objective lens, it can be divided into: ①Low-power objective refers to 1×-6×, NA value is 0.04-0.15; ②Medium-power objective refers to 6×-25×, NA value is 0.15-0.40; ③High-power objective refers to 25 ×—63×, NA value is 0.35—0.95; ④ Oil immersion objective refers to 90×—100×, NA value is 1.25—1.40. According to the degree of aberration correction, the classification can be divided into: ① Achromatic objective lens is a commonly used objective lens, marked with "Ach" on the shell, this objective lens can remove the chromatic aberration formed by red light and cyan. Light. It is often used in conjunction with Huygens eyepieces in microscopy. ②The apochromatic objective is marked with the word "Apo" on the objective housing. In addition to correcting the chromatic aberration of red, blue, and green light, it can also correct the phase difference caused by yellow light. It is often used in conjunction with compensating eyepieces. ③ Special objective lenses are manufactured on the basis of the above objective lenses to achieve a certain specific observation effect. Such as: objective lens with correction ring, objective lens with field diaphragm, phase contrast objective lens, fluorescence objective lens, strain-free objective lens, capless objective lens, long working distance objective lens, etc. The commonly used objective lenses in the current research are: semi-apochromatic objective (FL), plan objective (Plan), plan apochromatic objective (Plan Apo), super plan objective (Splan, super plan apochromat) objective (Splan) Apo), etc.
(4) Eyepiece The function of the eyepiece is to magnify the real image enlarged by the objective lens again, and reflect the object image to the eyes of the observer. The structure of the eyepiece is simpler than that of the objective lens. The eyepiece of an ordinary optical microscope usually consists of two lenses. The upper lens is called the "eyepiece" and the lower lens is called the "field lens". Between the upper and lower lenses or below the two lenses, there is a metal annular diaphragm or "field diaphragm". After magnification, the intermediate image of the objective lens falls on the plane of the field diaphragm, so an eyepiece micrometer can be placed. Commonly used eyepieces in optical microscopes are For Huygens eyepieces, if you need to conduct research, generally choose eyepieces with better performance, such as compensating eyepieces (K), flat eyepieces (P), and wide field eyepieces (WF). Use a photographic eyepiece (NFK) when taking pictures.
(2) Optical microscope The magnification of the microscope is done through the lens, and the imaging of a single lens has aberrations, which affect the imaging quality. A lens group consisting of a single lens is equivalent to a convex lens with better magnification. Figure 1-4 is the principle mode of microscope imaging. AB is the specimen.
(3) The performance of the microscope. The resolution of a microscope depends on various conditions of the optical system. The object being observed must have high magnification and be clear. Whether an object can show a clear and fine structure after magnification depends first on the performance of the objective lens, followed by the performance of the eyepiece and condenser.
1. Numerical aperture is also called aperture ratio (or aperture ratio), abbreviated as NA, and their values are marked on the objective lens and condenser lens. Aperture and numerical aperture are the main parameters of objective lenses and condensers, and are also important indicators for judging their performance. Numerical aperture is closely related to various properties of microscopes. It is proportional to the resolution of the microscope and inversely proportional to the depth of focus. It is proportional to the square root of the brightness of the mirror image. The numerical aperture can be expressed by the following formula: NA=n.sin α 2 where: n——the medium resolution between the objective lens and the sample α——the lens opening angle of the objective lens The so-called lens opening angle refers to the distance from the optical axis of the objective lens The angle between the light emitted by the upper object point and the edge of the effective diameter of the front lens of the objective lens is shown in Figure 1-5. The lens opening angle α is always less than 180°. Since the refractive index of air is 1, the numerical aperture of the dry objective is always less than 1, generally 0.05-0.95; if the oil immersion objective is immersed in cedar oil (with a refractive index of 1.515), the numerical aperture can reach 1.5. While theoretically the limit of numerical aperture is equal to the refractive index of the immersion medium used, in practice, it is impossible to reach this limit from a lens manufacturing technology perspective. Usually within the practical range, the largest numerical aperture of oil immersion objectives is 1.4. The medium refractive indices of several substances are as follows: 1.0 for air, 1.33 for water, 1.5 for glass, 1.47 for glycerin, and 1.52 for cedar. The effect of the refractive index of the medium on the optical path of the objective lens is shown in Figure 1-6.
2. The resolution D can be expressed by the following formula: D=λ/2N.A. The wavelength of visible light is 0.4-0.7 microns, with an average wavelength of 0.55 microns. If an objective with a numerical aperture of 0.65 is used, then D = 0.55 microns / 2 x 0.65 = 0.42 microns. This means that objects larger than 0.42 microns can be observed and objects smaller than 0.42 microns cannot be seen. If an objective with a numerical aperture of 1.25 is used, then D=2.20 microns. Any object to be inspected whose length is greater than this value will be visible. It can be seen that the smaller the D value, the higher the resolution and the clearer the object image. According to the above formula, the resolution can be improved by: (1) reducing the wavelength; (2) increasing the refractive index; (3) increasing the lens angle. Ultraviolet light-based microscopes and electron microscopes use short wavelengths of light to improve resolution to examine smaller objects. The resolution of the objective lens is closely related to the sharpness of the image. Eyepieces do not have this capability. The eyepiece only magnifies the image produced by the objective.
3. Magnification: The microscope magnifies the object, first through the objective lens * secondary magnification, and the eyepiece causes secondary magnification at the distance of bright vision. The magnification is the volume ratio of the back image to the original object. Therefore, the magnification (V) of the microscope is equal to the product of the magnification of the objective lens (V1) and the magnification of the eyepiece (V2), namely: V=V1×V2 The calculation method of the comparison can be obtained from the following formula M= △ × D F1 F2 F1 =Objective focal length F2=Eyepiece focal length △=Light pipe length D=Clear sight distance (=250mm) △=Magnification objective D=Eyepiece magnification M=Microscope magnification F1 F2 Setting △=160mm F1=4mm D=250mm F2=150mm Then M= △ × D= 160 × 250 =40×16.7=668 times F1 F2 4 15
4. Depth of focus: Observe the specimen under a microscope. When the focus is on a certain image plane, the image of the object is clear, and the image plane is the target plane. In addition to the target surface in the field of view, blurred object images can also be seen above and below the target surface. The distance between these two surfaces is called the depth of focus. The depth of focus of an objective is inversely proportional to the numerical aperture and magnification: the larger the numerical aperture and magnification, the smaller the depth of focus. Therefore, the adjustment of the oil mirror must be more careful than the adjustment of the low-power mirror, otherwise it is easy to cause the object to slip through and not be found.
