Microscope principle

The structure of ordinary optical microscope is mainly divided into three parts: mechanical part, lighting part and optical part.

1. Mechanical components

(1) mirror base: it is the base for the microscope to support the whole mirror body.

(2) Mirror column: it is an upright part above the mirror base, which is used to connect the mirror base and the mirror arm.

(3) Mirror arm: one end is connected to the lens column, and the other end is connected to the lens barrel, which is the hand-held part when taking and placing the microscope.

(4) Lens barrel: connected to the front upper part of the lens arm, with an eyepiece at the upper end and an objective lens converter at the lower end.

(5) The objective lens converter (rotator) is connected to the lower part of the prism housing and can rotate freely. There are 3-4 round holes on the disc, which is where the objective lens is installed. By rotating the converter, the objective lens with different multiples can be replaced. When you hear a knock, you can observe it. At this time, the optical axis of the objective lens is just aligned with the center of the aperture, and the optical path is connected.

(6) stage (stage): There are two shapes under the lens barrel, square and round, and there is a light hole in the center. The microscope we use has a slide specimen pusher (slide pusher) on the stage, a spring clip on the left side of the pusher is used to clamp the slide specimen, and a propeller adjusting wheel is arranged below the stage, which can make the slide specimen move back and forth left and right.

(7) Regulator: There are two kinds of screws installed on the mirror column, which make the mirror table move up and down during adjustment.

① coarse adjuster (coarse adjustment screw): The large screw is called coarse adjuster, which can make the stage move quickly and greatly, so the distance between the objective lens and the specimen can be adjusted quickly, so that the object image appears in the field of vision. Usually, when using a low-power lens, you can quickly find the object image by using the coarse tuner first.

② Fine adjuster (thin screw): Small screw is called fine adjuster, which can make the stage slowly rise and fall when moving. It is mostly used when using high-power lens to obtain clearer images of objects and observe the structures of specimens at different levels and depths.

2. Lighting part

It is installed under the mirror stage and comprises a reflector and a light collector.

(1) reflector: installed on the mirror base and can rotate in any direction. It has flat and concave sides, and its function is to reflect the light emitted by the light source to the condenser, and then illuminate the specimen through the light hole. Concave mirror has a strong condensing effect and is suitable for use in weak light, while the flat mirror has a weak condensing effect and is suitable for use in strong light.

(2) The light collector (condenser) is located on the light collector frame below the stage and consists of a condenser and an aperture. Its function is to focus light on the sample to be observed.

(1) condenser: It consists of one or several lenses, which can collect light, enhance the illumination of the specimen, and make the light enter the objective lens. There is an adjusting screw next to the lens column, which can rotate to lift the condenser and adjust the brightness of light in the field of view.

(2) Aperture (iridescent aperture): Below the condenser, it consists of more than a dozen pieces of metal, and a handle extends out. Press it to adjust the size of the opening, thus adjusting the amount of light.

3. Optical part

(1) spectacle lens: it is installed at the upper end of the lens barrel, and usually has 2-3 eyepieces, on which symbols of 5×, 10× or 15× are engraved to indicate its magnification. General installation 10× eyepiece.

(2) Objective lens: installed on the rotating body at the lower end of the lens barrel, there are generally 3-4 objective lenses, of which the shortest one engraved with the symbol "10X" is a low-power mirror, the longer one engraved with the symbol "40X" is a high-power mirror, and the longest one engraved with the symbol "10x" is an oil mirror. Besides,

The magnification of microscope is the product of the magnification of objective lens and the magnification of eyepiece. For example, if the objective lens is 10× the eyepiece is 10×, the magnification is10×/kloc-0 =100.

Microscopic imaging principle

When the object to be observed is placed outside the focus of the objective lens near the focus, the real image formed behind the objective lens is just in the focus of the eyepiece near the focus, and it is enlarged into a virtual image again through the eyepiece. What is observed is the inverted image virtual image after two enlargements.

Classification of microscope

Microscope can be divided into optical microscope and electron microscope.

1590, Zhan Sen and his son in the Netherlands first invented the optical microscope. At present, the optical microscope can enlarge the object by 1500 times, and the minimum resolution is 0.2 micron. There are many kinds of optical microscopes, except general ones, mainly dark-field microscopes, which have a dark-field condenser, so that the illumination beam does not enter from the central part, but shines on the specimen from the periphery. The fluorescence microscope uses ultraviolet light as the light source to make the irradiated object emit fluorescence.

The electron microscope was first assembled by Noel and Ha Roska in Berlin in 193 1. This microscope uses high-speed electron beams instead of light beams. Because the wavelength of electron flow is much shorter than that of light wave, the magnification of electron microscope can reach 800 thousand times, and the lowest resolution limit is 0.2 nm. 1963 uses a scanning electron microscope, which enables people to see the tiny structures on the surface of objects.

According to the illumination method, there are two types of optical microscopes: transmission type and reflection (descending) type. Transmission microscope is an illumination method that uses transmission illumination to penetrate transparent objects. Reflective microscope illuminates opaque objects from above the objective lens. Another classification method is divided into bright field microscope, dark field microscope, phase contrast microscope, polarizing microscope, interference phase contrast microscope and fluorescence microscope according to different observation methods. Each microscope generally has two types: transmission type and reflection type. Among these microscopes, especially the bright field microscope is the most basic foundation of all microscopes. The transmission (absorption) and reflectivity of objects observed by this microscope are different in different places. This kind of object is called amplitude object, which varies with the intensity (amplitude) of illumination light. Colorless and transparent objects can only be observed by naked eyes when the illumination phase changes. Because bright field microscope can't change the phase, it can't be observed for transparent and undyed samples.

The purpose of optical microscope is to make the specimen image unclear to the naked eye. People imagine that through a device, the naked eye can observe the shape of specimen tissues and the structure between them. This imaginary device was created by later generations. At present, it is widely used in the observation, determination, analysis, classification and identification of various tiny objects. The wavelength range is not limited to visible light band (4000~7000), but also includes (> 2000) to infrared (1~2u), as well as eye observation, microscope, photography and general radiation detector amplification.

The comprehensive magnification of the microscope is the product of the objective lens magnification G 1 and the eyepiece magnification G2, where G = G=G 1×G2. G 1 is 1~ 100 times, G2 is 5~20 times.

Numerical aperture) N.A is the basic data that determines the resolution, focal depth and image brightness of the objective lens. When the focus of the objective lens is aligned, the included angle between the outermost oblique light of the front lens of the objective lens and the optical axis of the microscope is α, that is, if the half aperture angle of the objective lens is n, then N.A. = n× sin α.

N is usually 1 in air. When water, glycerin and grease are immersed between the objective lens and the sample, the refractive index of the sample changes with the immersion liquid. This objective lens is called immersion lens; If it is air, it is called a dry objective.

On the microscope, the device to limit the field of view is the field stop. When the field stop is viewed from the objective lens side, the diameter value in mm is called the field number. Actual field of vision = field of vision.

Actual field of view = number of fields of view/objective magnification

For example, if the number of fields of view is 20, then the 10× objective lens will observe a field of view of 2mm. When the condenser is used, the N.A. value of the condenser is determined according to the variable field of view diaphragm, which is determined by the variable aperture diaphragm of the condenser.

The invention of microscope makes people see many creatures that have never been seen before, such as bacteria and viruses, and also makes people see many tiny structures of organisms, such as mitochondria, which plays an important role in promoting the development of organisms. Microscope is one of the important instruments in biological research. Microscope also has important applications in medicine, industry and agriculture, such as counting red blood cells in human blood in medical diagnosis.

/kloc-in the mid-9th century, people invented the optical microscope.

1665, British scholar robert hooke designed and manufactured the first optical microscope with a magnification of 40~ 140, and used it to observe and describe plant cells for the first time. In the same year, he published the book Microatlas.

Since then, A. V .Leeuwenhoek, a Dutch scholar, has observed animal cells with a more advanced microscope designed by himself and described the morphology of the nucleus. Up to now, optical microscope has developed from ordinary compound optical microscope to fluorescence microscope, confocal laser scanning microscope, digital imaging microscope, dark field microscope, phase difference and differential interference microscope, video recording and contrast microscope.

It can be seen that optical microscopy has become an essential tool for human beings to understand the micro-world. With it, people can understand cells. However, accurate theoretical calculation shows that the quality of the optical microscope has not improved-no matter how many lenses are used, the magnification is the most 1000~ 1500 and the resolution is the most. This has become a bottleneck for human beings to understand smaller objects: viruses, molecules and atoms.

Helen Hall, a famous physicist, proved theoretically that the factor limiting the resolution and magnification of optical microscope is the wavelength of light. Therefore, people turn to find an imaging medium-wave, which has the characteristics of visibility, photographing, short wave length, and being able to change the motion route of the wave with devices.

At the beginning of the 20th century, Chayes invented the ultraviolet microscope, which greatly improved the resolution. This is a qualitative leap, but ultraviolet is still not the best imaging medium and cannot meet the needs of scientific research and production.

Bosch, a German scientist, pointed out in 1926 that the axisymmetric magnetic field acts as a lens for the electron beam. Unfortunately, researchers did not consider using it to enlarge objects.

1932, Luska and Knohl, young researchers in the pressure laboratory of Berlin University of Science and Technology, made some improvements to the cathode ray oscilloscope, and successfully obtained images of the copper mesh after being magnified several times, which greatly inspired people and established the electron microscope method.

At the end of 1933, Luska made an electron microscope with a magnification of 10,000 times, and took magnified images of metal foil and fiber. The magnification of electron microscope exceeds that of optical microscope.

1937, Clausse and Mueller of Berlin Technical University successfully manufactured an electron microscope with a resolution of nanometer (10-9m). When Siemens learned about it, it turned its main energy to manufacturing a suitable electron microscope and hired Luce to conduct research. The next year, the resolution of the first batch of electron microscopes reached.

Subsequently, under the research of people, the quality of electron microscope has been continuously improved. Today, its resolution and magnification enable people to know viruses, molecules, atoms and quarks more accurately.

ultramicroscope

Because the dark field microscope does not inject transparent light into the direct observation system, the field of view is dark when there is no object, so it is impossible to observe any object. When there is an object, the diffracted light and scattered light of the object are bright and visible in the dark background. When observing an object in a dark field, most of the illumination light is reflected back. Because of the different position, structure and thickness of the object (specimen), the scattering and refraction of light have great changes.

phase contrast microscope

Structure of phase contrast microscope;

The phase contrast microscope is a microscope that uses the phase contrast method. Therefore, the microscope should add the following accessories:

(1) objective lens with phase plate (phase annular plate), phase difference objective lens.

(2) A condenser with a phase ring (annular slit plate) and a phase difference condenser.

(3) Monochrome filter-(green).

Performance description of various components

(1) The phase plate shifts the phase of direct light by 90 degrees, absorbing and weakening the intensity of light. The brightness of the phase plate must be ensured when it is installed in the appropriate position of the back focal plane of the objective lens. In order to reduce the influence of diffracted light, the phase plate is made into a ring shape.

(2) The phase ring (annular aperture) can be replaced by a turntable according to the magnification of each objective lens.

(3) The monochromatic filter is a green filter with a central wavelength of 546nm (nanometer). Usually monochromatic filters are used for observation. The phase plate moves 90 degrees at a specific wavelength to see the phase of the direct light. When a specific wavelength is needed, the appropriate filter must be selected, and the contrast will be improved after inserting the filter. In addition, the center of the phase ring seam must be adjusted to the correct orientation to operate, and the centering telescope is the component that plays this role.

Video microscope

The traditional microscope combines a camera system, a display or a computer to achieve the purpose of magnifying and observing the measured object.

The earliest prototype should be a camera microscope. Through the principle of pinhole imaging, the images obtained under the microscope are projected onto the photosensitive photos, so as to get the picture. Or directly dock the camera with the microscope to take pictures. With the rise of CCD camera, microscope can transmit real-time images to TV or monitor for direct observation, or shoot them through camera. In the mid-1980s, with the development of digital industry and computer industry, the function of microscope has been improved through them, making it simpler and easier to operate. By the end of 1990s, with the development of the semiconductor industry, the wafer demanded that the microscope could bring more coordination functions. The combination of hardware and software makes the microscope more intelligent and humanized, and makes the microscope have a greater development in industry.

fluorescent microscope

In the fluorescence microscope, excitation light with a specific wavelength must be selected from the illumination light of the sample to generate fluorescence, and then the fluorescence must be separated from the mixed light of excitation light and fluorescence for observation. Therefore, the filter system plays an extremely important role in the selection of specific wavelengths.

The principle of fluorescence microscope:

(a) Light source: A light source emits light of various wavelengths (from ultraviolet to infrared).

(b) Excitation filter light source: transmits light with a specific wavelength that can make the sample emit fluorescence, while blocking light that is useless for excitation fluorescence.

(c) Fluorescent specimens: generally stained with fluorescent pigments.

(d) Blocking filter: blocking the excitation light that is not absorbed by the specimen, so that the fluorescence is selectively transmitted, and some wavelengths in the fluorescence are also selectively transmitted.

Micrometer polarizer

Polarizing microscope is a kind of microscope used to study so-called transparent and opaque anisotropic materials. Under the polarizing microscope, all substances with birefringence can be clearly distinguished. Of course, these substances can also be observed by dyeing, but some of them are impossible, and a polarizing microscope must be used.

Characteristics of (1) polarizing microscope

A method of changing ordinary light into polarized light, which is used for microscopic examination to identify whether a substance is single refraction (isotropic equivalence) or birefringence (anisotropy). Birefringence is the basic characteristic of crystals. Therefore, polarizing microscope is widely used in the fields of minerals, chemistry, biology and botany.

(2) The basic principle of polarizing microscope

The principle of polarizing microscope is complicated, so I won't introduce it too much here. Polarizing microscope must have the following accessories: polarizer, analyzer, compensator or phase plate, special stress-free objective lens and rotary stage.

ultrasonic microscope

The characteristic of ultrasonic scanning microscope is that it can accurately reflect the interaction between sound waves and the elastic medium of tiny samples, and analyze the signals fed back from inside the samples! Each pixel in the image (C-scan) corresponds to a signal fed back by a two-dimensional coordinate point at a certain depth in the sample. Z. A sensor with good focusing function can simultaneously transmit and receive acoustic signals. Therefore, a complete image is formed by scanning samples point by point and line by line. The reflected ultrasonic wave plus positive or negative amplitude makes the depth of the sample can be reflected by the time of signal transmission. The digital waveform on the user's screen shows the received feedback information (A scan). Set the corresponding gate circuit, with this quantitative time difference measurement (feedback time display), you can choose the depth of the sample you want to observe.

dissecting microscope

Dissecting microscope, also known as solid microscope or stereoscopic microscope, is a microscope designed for different work requirements. When observed by dissecting microscope, the light entering the eyes comes from an independent light path, and these two light paths have only a small angle, so the sample can present a three-dimensional appearance when observed. There are two kinds of optical path designs in dissecting microscope: Greenough type and telescope type. Dissecting microscope is often used for surface observation of some solid samples, or for dissection, clock making and small circuit board inspection.