optical microscopy

microscope mainly includes four parts: objective lens, eyepiece, reflector and condenser. Broadly speaking, it also includes illumination light sources, filters, cover slips, slides, etc.

(I), objective lens

objective lens is an important part of zui that determines the performance of the microscope. It is installed on the objective lens converter and is close to the object to be observed, so it is called the objective lens or the objective lens.

1. Classification of objective lens

objective according to the different conditions of use can be divided into dry objective and immersion objective; which immersion objective can be divided into water immersion objective and oil immersion objective (commonly used magnification is 90-100 times).

According to the different magnification can be divided into low-power objective (10 times below), medium-power objective (about 20 times) and high-power objective (40-65 times).

According to the aberration correction, it is divided into achromatic objective lens (commonly used, the objective lens that can correct the chromatic aberration of two kinds of color light in the spectrum) and complex chromatic objective lens (the objective lens that can correct the chromatic aberration of three kinds of color light in the spectrum, which is expensive and less used).

2, the main parameters of the objective lens:

objective include: magnification, numerical aperture and working distance.

①. Magnification refers to the ratio of the size of the image seen by the eye to the size of the corresponding specimen. It refers to the ratio of length rather than the ratio of area. For example, the magnification is 100 ×, which refers to the specimen with a length of 1μm. The length of the magnified image is 100 μm, which is 10,000 times larger if calculated by area.

microscope is equal to the product of the objective and eyepiece magnifications.

②. Numerical aperture is also called lens aperture ratio, abbreviated NA or A, which is the main parameter of objective lens and condenser and is proportional to the resolution of microscope. The numerical aperture of the dry objective lens is 0.05-0.95, and that of the oil immersion objective lens (cedar oil) is 1.25.

3. The working distance refers to the distance from below the front lens of the objective lens to above the cover glass of the specimen when the observed specimen zui is clear. The working distance of the objective lens is related to the focal length of the objective lens. The longer the focal length of the objective lens, the lower the magnification and the longer the working distance. For example, the 10x objective lens is marked with 10/0.25 and 160/0.17, where 10 is the magnification of the objective lens; 0.25 is the numerical aperture; 160 is the length of the barrel (in mm);0.17 is the standard thickness of the cover glass (in mm). The effective working distance of the 10x objective lens is 6.5mm, and the effective working distance of the 40x objective lens is 0.48mm.

3. The function of the objective lens is to magnify the specimen * times. It is an important component of zui that determines the performance of the microscope-the resolution.

resolution is also called resolution or resolution. The size of the resolution is expressed by the value of the resolution distance (the zui small distance between the two object points that can be resolved). At the photopic distance (25cm), the normal human eye can see two object points 0.073mm apart, and this value of 0.073mm is the resolution distance of the normal human eye. The smaller the resolving distance of the microscope, the higher its resolving power, that is, the better its performance.

microscope is determined by the resolving power of the objective lens, which in turn is determined by its numerical aperture and the wavelength of the illuminating light.

When using ordinary central illumination (bright-view illumination that allows light to pass uniformly through the specimen), the resolution distance of the microscope is d = 0.61 & lambda;/NA

.

& lambda;– wavelength of illumination light, unit: nm.

NA-numerical aperture of the objective lens

, for example, an oil immersion objective has a numerical aperture of 1.25 nm and a visible light wavelength range of 400-700nm, taking its average wavelength of 550 nm, d = 270 nm, which is approximately equal to half the wavelength of the illumination light. In general, the limit of the resolving power of a microscope illuminated with visible light is 0.2 μm.

(II), eyepiece

is also called eyepiece because it is close to the observer’s eye. Mounted on the upper end of the lens barrel.

1. Structure of the eyepiece

usually the eyepiece consists of two sets of lenses, the upper lens is called the eye lens, and the lower lens is called the converging lens or field lens. A diaphragm is installed between the upper and lower lenses or under the field lens (its size determines the size of the field of view). Because the specimen is imaged just on the diaphragm plane, a small piece of hair can be glued to this diaphragm as a pointer to indicate a target with a certain characteristic. An eyepiece micrometer can also be placed on it to measure the size of the specimen being observed.

eyepiece, the greater the magnification (because the magnification of the eyepiece is inversely proportional to the focal length of the eyepiece).

2. The role of the eyepiece

is to further enlarge the real image that has been magnified by the objective lens to the extent that the human eye can easily distinguish it clearly.

common eyepiece is 5-16 times.

3. The relationship between the eyepiece and the objective lens

the fine structure of the objective lens has been clearly distinguished, if it is not magnified by the eyepiece and cannot reach the size that the human eye can distinguish, it cannot be seen clearly. However, the fine structure that the objective lens cannot distinguish is still not clear even though it is magnified by the high-power eyepiece, so the eyepiece can only play a magnifying role and will not improve the resolution of the microscope. Sometimes, although the objective lens can distinguish two close object points, it is still unable to see clearly because the distance between the images of the two object points is less than the resolution distance of the eyes. Therefore, the eyepiece and the objective lens are each other and restrict each other.

(III), concentrator

concentrator is also called collector. Located on a concentrator holder below the specimen. It is mainly composed of a condenser and an iris diaphragm. Among them, the condenser can be divided into a bright field condenser (common microscope configuration) and a dark field condenser.

1. Main parameters of light mirror

is the main parameter of the condenser lens. The large numerical aperture of zui is generally 1.2-1.4, and the numerical aperture has a certain variable range. Usually, the number engraved on the upper lens frame represents the large numerical aperture of zui. By adjusting the opening degree of the lower iris, various numerical apertures below this number can be obtained to meet the needs of different objective lenses. Some condenser lenses are composed of several groups of lenses. A group of lenses above zui can be removed or removed from the optical path to make the numerical aperture of the condenser smaller to adapt to the illumination of low-power objective observation.

2, the role of the condenser

condenser is equivalent to a convex lens, which converges light to enhance the illumination of the specimen. The condensing focal point of the condenser is generally designed to be about 1.25mm above the lens plane at its upper end. (The focus is on the specimen to be observed, and the thickness of the slide is about 1.1mm)

3. Variable aperture

iris diaphragm is also called aperture, located at the bottom of the condenser, composed of more than a dozen pieces of metal sheet, the central part of the formation of a round hole. Its function is to adjust the light intensity and to adapt the numerical aperture of the condenser lens to the numerical aperture of the objective lens. The larger the variable aperture is, the larger the numerical aperture is (after observation, the aperture should be adjusted to zui).

under the iris, there is also a circular filter carrier.

Description: In the middle school laboratory, only teachers’ microscopes (1600 × or 1500 ×) are equipped with concentrators, while students’ microscopes (640 × or 500 ×) are equipped with rotating light bars. A disc that is tightly attached to the stage and can rotate in a circle and rotate the light bar (also called a shutter). There are round holes of different sizes on the light bar, called apertures. The diameters are 2, 3, 6, 12 and 16mm respectively. Rotate the rotating light bar. Each aperture on the light bar can be aligned with the light passing hole, and the intensity of light can be adjusted through apertures of different sizes.

(IV) mirror

reflector is a double-sided mirror that can be rotated at will. Its diameter is 50mm, one side is flat and one side is concave. Its function is to reflect the light from any direction through the light hole. The ability of the plane mirror to reflect light is weak, and it is used when the light is strong, and the ability of the concave mirror to reflect light is strong, and it is used when the light is weak.

reflector is usually a plane mirror on one side and a concave mirror on the other, which is installed under the condenser and can be rotated at will in both horizontal and vertical directions.

reflector is to direct the light emitted by the light source or the natural light toward the condenser. When using a condenser, a plane mirror is generally used, and a concave mirror is not used; when the light is strong, a plane mirror is used, and a concave mirror is used when the light is weak.

After observation, the mirror should be placed vertically.

(V) lighting source

microscope can be used with natural or artificial light sources

1. Natural light source

light comes from the sky, zui is reflected by white clouds. Do not use direct sunlight.

2. Artificial light source

①, the basic requirements of artificial light source: have sufficient luminous intensity; light source heat can not be too much.

②. Commonly used artificial light source: microscope lamp; fluorescent lamp

(VI) filter

is installed between the light source and the condenser. The role is to let the selected band of light through, and absorb other light, that is, in order to change the spectral components of light or weaken the intensity of light. Divided into two categories: filters and liquid filters.

(VII) coverslips and slides

The surface of the coverslip and slide should be fairly flat, free of bubbles and scratches. Zui good choice colorless, good transparency, should be washed before use.

cover glass is 0.17±0.02mm. If the cover glass is not used or the thickness of the cover glass is not suitable, the imaging quality will be affected.

glass slide is 1.1±0.04mm, and the general usable range is 1-1.2mm. If it is too thick, it will affect the efficiency of the concentrator, and if it is too thin, it will easily break.