Item h placed slightly further than the front focus of the lens. The lens gives real, inverse, augmented image H’ , located between the front focus of the eyepiece and the optical center of the eyepiece. This intermediate image is viewed through the eyepiece as if through a magnifying glass. The eyepiece gives imaginary, direct, magnified image H, which is located at the distance of best vision S ≈ 25 cm from the optical center of the eye.
We look at this image with our eyes and it forms on its retina. real, inverse, reduced image.
Microscope Magnification– the ratio of the dimensions of the virtual image to the dimensions of the object viewed through the microscope: 
. Multiply the numerator and denominator by the size of the intermediate image H’
:
. Thus, the magnification of the microscope is equal to the product of the objective magnification and the eyepiece magnification. Lens magnification can be expressed in terms of the characteristics of the microscope using the similarity of right triangles 
, Where L
optical
tube length: the distance between the back focus of the lens and the front focus of the eyepiece (we assume that L
>> F about). Eyepiece magnification
. Therefore, the magnification of the microscope is: 
.
4. Resolution and resolution limit of the microscope. Diffraction phenomena in a microscope, the concept of Abbe's theory.
Microscope resolution limitz - this is the smallest distance between two points of an object viewed through a microscope, when these points are still perceived separately. The resolution limit of a conventional biological microscope lies in the range of 3-4 microns. Resolution microscope is the ability to provide a separate image of two closely located points of the object under study, that is, this is the reciprocal of the resolution limit.
Diffraction of light places a limit on the ability to distinguish the details of objects when they are observed through a microscope. Since light does not propagate rectilinearly, but bends around obstacles (in this case, the objects in question), images of small details of objects turn out blurry.
E. Abbe suggested diffraction theory of microscope resolution. Let the object that we want to examine through a microscope be a diffraction grating with a period d. Then the minimum detail of the object that we must distinguish will be precisely the lattice period. Light diffraction occurs on the grating, but the diameter of the microscope objective is limited, and at large diffraction angles, not all the light passing through the grating enters the objective. In reality, light from an object propagates towards the lens in a certain cone. The resulting image is closer to the original, the more maxima involved in the formation of the image. Light from an object propagates to the lens from a condenser in the form of a cone, which is characterized by angular aperture
u- the angle at which the lens is visible from the center of the object under consideration, that is, the angle between the outer rays of the conical light beam entering the optical system. According to E. Abbe, to obtain an image of a grating, even the most fuzzy one, rays of any two orders of the diffraction pattern must enter the lens, for example, rays forming the central and at least the first diffraction maximum. Let us recall that for the oblique incidence of rays on a diffraction grating, its main formula has the form: . If the light comes at an angle 
, and the diffraction angle for first maximum equals 
, then the formula takes the form 
. The resolution limit of the microscope should be taken as the constant of the diffraction grating, then 
, where is the wavelength of light.
As can be seen from the formula, one way to reduce the resolution limit of a microscope is to use light with a shorter wavelength. In this regard, an ultraviolet microscope is used, in which microobjects are examined in ultraviolet rays. The basic optical design of such a microscope is similar to that of a conventional microscope. The main difference is the use of optical devices that are transparent to UV light and the image registration features. Since the eye does not perceive ultraviolet radiation (in addition, it burns the eyes, i.e. is dangerous for the organ of vision), photographic plates, fluorescent screens or electro-optical converters are used.
If a special liquid medium called immersion, then the resolution limit also decreases: 
, Where n– absolute refractive index of immersion, A
– lens numerical aperture. Water is used as immersion ( n
=
1.33), cedar oil ( n= 1.515), monobromonaphthalene ( n
=
1.66), etc. For each type of immersion, a special lens is made, and it can only be used with this type of immersion.
Another way to reduce the resolution limit of a microscope is to increase the aperture angle. This angle depends on the size of the lens and the distance from the subject to the lens. However, the distance from the object to the lens cannot be changed arbitrarily; it is constant for each lens and the object cannot be brought closer. In modern microscopes, the aperture angle reaches 140 o (respectively, u/2 = 70 o). With this angle, maximum numerical apertures and minimum resolution limits are obtained.
The data is given for an oblique incidence of light on an object and a wavelength of 555 nm, to which the human eye is most sensitive.
Please note that the eyepiece does not affect the resolution of the microscope at all, it only creates a magnified image of the lens.
It is technically possible to create optical microscopes whose lenses and eyepieces will provide a total magnification of 1500-2000 or more. However, this is impractical, since the ability to distinguish small details of an object is limited by diffraction phenomena. As a result, the image of the smallest details of an object loses sharpness, a violation of the geometric similarity of the image and the object may occur, neighboring points will merge into one, and the image may completely disappear. Therefore, in optics there are the following concepts that characterize the quality of a microscope:
Microscope resolution- the property of a microscope to provide a separate image of small details of the object under consideration.
Resolution limit- this is the smallest distance between two points that are visible separately in a microscope.
The lower the resolution limit, the higher the resolution of the microscope!
The resolution limit determines the smallest size of details that can be distinguished in a specimen using a microscope.
The theory of the resolving power of the microscope was developed by the director of the K. Zeiss plant in Jena, professor of optics E. Abbe (1840-1905). As a simple microscopic specimen, he took a diffraction grating (Fig. 2), studied the mechanism of image formation in a microscope and showed the following.
Let's introduce the concept aperture angle- this is the angle between the outer rays of a conical light beam coming from the middle of the object into the lens (Fig. 3, A). To create an image, that is, to resolve an object, it is enough for the lens to receive rays that form only zero- and first-order maxima on at least one side (Fig. 2 and 3, b). The participation of rays from a larger number of maxima in the formation of the image increases the quality of the image and its contrast. Therefore, the rays that form these maxima must be within the aperture angle of the lens.
 |
a) b) c) d)
1 - front lens, 2 - lens
Thus, if the object is a diffraction grating with a period d and the light falls on it normally (Fig. 2 and 3, b), then the rays forming maxima of the zero and first order on both sides must necessarily participate in the formation of the image, and the angle j 1 is the angle of deviation of the rays forming the maximum of the first order; accordingly, in extreme cases, it must be equal to the angle U/2.
If we take a lattice with a smaller period d’, then angle j’ 1 will be greater than angle U/2 and the image will not appear. This means the lattice period d can be taken as the resolution limit of the microscope Z. Then, using the diffraction grating formula, we write for k=1:
Replacing d on Z, and j 1 on U/2, we get
. (6)
During microscopy, light rays strike an object at different angles. With oblique incidence of rays (Fig. 3, G) the resolution limit decreases, since only rays that form zero-order and first-order maxima on one side will participate in image formation, and the angle j 1 will be equal to the aperture angle U. Calculations show that the formula for the resolution limit in this case takes the following form:
. (7)
If the space between the object and the lens is filled with an immersion medium with a refractive index n, which is greater than the refractive index of air, then the wavelength of light l n= l ¤ n. Substituting this expression into the formula for the resolution limit (7), we obtain
, or 
. (8)
Thus, formula (7) determines the resolution limit for a microscope with a dry objective, and formula (8) for a microscope with an immersion objective. Values sin 0.5 U And n× sin0.5 U in these formulas is called the numerical aperture of the lens and is designated by the letter A. Taking this into account, the formula for the resolution limit of a microscope in general form is written as follows:
As can be seen from formulas (8) and (9), the resolution of the microscope depends on the wavelength of light, the aperture angle, the refractive index of the medium between the lens and the object, the angle of incidence of light rays on the object, but it does not depend on the parameters of the eyepiece. The eyepiece does not provide any additional information about the structure of the object, does not improve image quality, it only enlarges the intermediate image.
The resolution of a microscope can be increased by using immersion and reducing the wavelength of light. The increase in resolution when using immersion can be explained as follows. If there is air between the lens and the object (dry lens), then the light ray, when passing from the cover glass into air, a medium with a lower refractive index, significantly changes its direction as a result of refraction, so fewer rays enter the lens. When using an immersion medium, the refractive index of which is approximately equal to the refractive index of glass, no change in the path of rays in the medium is observed and more rays enter the lens.
Water is used as an immersion liquid ( n=1.33), cedar oil ( n=1.515), etc. If the maximum aperture angle for modern lenses reaches 140 0, then for a dry lens A=0.94, and for a lens with oil immersion A=1.43. If in the calculation we use the light wavelength l = 555 nm, to which the eye is most sensitive, then the resolution limit of a dry lens will be 0.30 µm, and with oil immersion - 0.19 µm. The numerical aperture value is indicated on the lens barrel: 0.20; 0.40; 0.65, etc.
Increasing the resolution of an optical microscope by reducing the wavelength of light is achieved by using ultraviolet radiation. For this purpose, there are special ultraviolet microscopes with quartz optics and devices for observing and photographing objects. Since these microscopes use light with a wavelength approximately half that of visible light, they are capable of resolving drug structures with dimensions of about 0.1 μm. Ultraviolet microscopy has another advantage - it can be used to examine unstained preparations. Most biological objects are transparent in visible light because they do not absorb it. However, they have selective absorption in the ultraviolet region and are therefore easily visible under ultraviolet rays.
An electron microscope has the highest resolution, since the wavelength of an electron moving is 1000 times less than the wavelength of light.
Useful microscope magnification limited by its resolving power and the resolving power of the eye.
The resolution of the eye is characterized by the smallest angle of view at which the human eye can still distinguish two points of an object separately. It is limited by diffraction on the pupil and the distance between the light-sensitive cells of the retina. For a normal eye, the smallest visual angle is 1 minute. If an object is at a distance of best vision - 25 cm, then this angle corresponds to an object measuring 70 microns. This value is considered the limit of resolution of the naked eye Zr at the best viewing distance. However, it has been shown that the optimal value Zr equal to 140-280 microns. In this case, the eye experiences the least strain.
Useful microscope magnification they call it the maximum magnification at which the eye is still able to distinguish details equal in magnitude to the resolution limit of the microscope.
The linear magnification of a microscope is equal to the ratio of the image size of an object located at the distance of best vision to the size of the object itself (see formula 1). If we take the resolution limit of the microscope as the size of the object Z, and for image size - the resolution limit of the naked eye at the distance of best vision Zr, then we get the formula for the useful magnification of a microscope:
Substituting into this formula Z from expression (9), we obtain
. (11)
Substituting into formula (11) a light wavelength of 555 nm (555×10 -9 m), the optimal values of the eye resolution limits are 140-280 µm (140-280×10 -6 m), we find the range of useful magnification values of the microscope
500 A < TO n< 1000 A .
For example, when using the best immersion objectives with a numerical aperture of 1.43, the useful magnification will be 700-1400, which shows that it is not practical to design optical microscopes with high magnification. However, at present, this issue has lost its urgency due to the widespread use in biology and medicine of an electron microscope, which provides an increase of up to 600,000 and a resolution limit of up to 0.1 nm.
Purpose of the work. Familiarization with the device of a microscope and determination of its resolution.
Devices and accessories: Microscope, metal plate with a small hole, lighting mirror, ruler with scale.
Introduction
A microscope consists of an objective and an eyepiece, which are complex lens systems. The path of rays in a microscope is shown in Fig. 1, in which the objective and eyepiece are represented by single lenses.
The object in question AB is placed slightly further from the main focus of the lens F about. The microscope lens gives a real, inverse and magnified image of the object (AB in Fig. 1), which is formed behind the double focal length of the lens. The magnified image is viewed by the eyepiece as a magnifying glass. The image of an object viewed through the eyepiece is virtual, inverse and magnified.
The distance between the back focus of the lens and the front focus of the eyepiece is called optical spacing of the system or optical tube length microscope .
The magnification of a microscope can be determined by the magnification of the objective and eyepiece:
N = N about N about = ───── (1)
f about f ok
where N about and N about are the magnification of the lens and eyepiece, respectively; D - distance of best vision for a normal eye (~25 cm); is the optical length of the microscope tube; f about and f OK- main focal lengths of the lens and eyepiece.
When analyzing formula (1), we can conclude that microscopes with high magnification can examine any small objects. However, the useful magnification provided by a microscope is limited by diffraction phenomena, which become noticeable when viewing objects whose dimensions are comparable to the wavelength of light.
Resolution limit microscope is the smallest distance between points, the image of which is obtained separately in the microscope.
According to Abbe's theory, the resolution limit of a microscope is determined by the expression:
d = ───── (2)
where d is the linear size of the object in question; - wavelength of the light used; n is the refractive index of the medium between the object and the lens; is the angle between the main optical axis of the microscope and the boundary ray (Fig. 2).
IN 
the quantity A = nsin is called numerical aperture of the lens
, and the reciprocal of d is microscope resolution
. From expression (2) it follows that the resolution of the microscope depends on the numerical aperture of the lens and the wavelength of light that illuminates the object in question.
If the object is in the air (n=1), then in the microscope it is possible to distinguish points of the object, the distance between which is:
d = ─────
For microscopic objects, the angle is close to 90 degrees, then sin 1, which means that objects located at a distance of ~ 0.61 from each other can be examined in a microscope. In the case of visual observations (the maximum sensitivity of the eye occurs in the green region of the visible spectrum 550 nm), objects located at a distance of ~300 nm can be seen in a microscope.
As follows from expression (2), the resolution of a microscope can be increased by reducing the wavelength of light that illuminates the object. Thus, when photographing objects in ultraviolet light (~ 250-300 nm), the resolution of the microscope can be doubled.
As you know, a person receives the bulk of information about the world around him through vision. The human eye is a complex and perfect device. This device created by nature works with light - electromagnetic radiation, the wavelength range of which is between 400 and 760 nanometers. The color that a person perceives changes from purple to red.
Electromagnetic waves corresponding to visible light interact with the electronic shells of atoms and molecules in the eye. The result of this interaction depends on the state of the electrons in these shells. Light can be absorbed, reflected or scattered. What exactly happened to the light can tell a lot about the atoms and molecules with which it interacted. The range of sizes of atoms and molecules is from 0.1 to tens of nanometers. This is many times shorter than the wavelength of light. However, objects of precisely this size - let's call them nanoobjects - are very important to see. What needs to be done for this? Let's first discuss what the human eye can see.
Usually, when talking about the resolution of a particular optical device, they operate with two concepts. One is angular resolution and the other is linear resolution. These concepts are interrelated. For example, for the human eye, the angular resolution is approximately 1 arc minute. In this case, the eye can distinguish two point objects located 25–30 cm away from it only when the distance between these objects is more than 0.075 mm. This is quite comparable to the resolution of a conventional computer scanner. In fact, 600 dpi resolution means the scanner can distinguish dots as close as 0.042 mm apart.
In order to be able to distinguish objects located at even smaller distances from each other, an optical microscope was invented - a device that increases the resolution of the eye. These devices look different (as can be seen from Figure 1), but their operating principle is the same. The optical microscope made it possible to push the resolution limit to fractions of a micron. Already 100 years ago, optical microscopy made it possible to study micron-sized objects. However, at the same time it became clear that it was impossible to achieve a further increase in resolution by simply increasing the number of lenses and improving their quality. The resolution of an optical microscope turned out to be limited by the properties of light itself, namely its wave nature.
At the end of the century before last, it was established that the resolution of an optical microscope is . In this formula, λ is the wavelength of light, and n sin u- the numerical aperture of the microscope lens, which characterizes both the microscope and the substance that is located between the object of study and the microscope lens closest to it. Indeed, the expression for the numerical aperture includes the refractive index n environment between the object and the lens, and the angle u between the optical axis of the lens and the outermost rays that exit the object and can enter that lens. The refractive index of vacuum is equal to unity. For air this indicator is very close to unity, for water it is 1.33303, and for special liquids used in microscopy to obtain maximum resolution, n reaches 1.78. Whatever the angle u, the value sin u cannot be more than one. Thus, the resolution of an optical microscope does not exceed a fraction of the wavelength of light.
The resolution is generally considered to be half the wavelength.
Intensity, resolution and magnification of an object are different things. You can make it so that the distance between the centers of images of objects that are located 10 nm from each other will be 1 mm. This would correspond to an increase of 100,000 times. However, it will not be possible to distinguish whether it is one object or two. The fact is that images of objects whose dimensions are very small compared to the wavelength of light will have the same shape and size, independent of the shape of the objects themselves. Such objects are called point objects - their sizes can be neglected. If such a point object glows, then an optical microscope will depict it as a light circle surrounded by light and dark rings. We will further, for simplicity, consider light sources. A typical image of a point light source obtained using an optical microscope is shown in Figure 2. The intensity of the light rings is much less than that of the circle and decreases with distance from the center of the image. Most often, only the first light ring is visible. The diameter of the first dark ring is . The function that describes this intensity distribution is called the point spread function. This function does not depend on what the magnification is. The image of several point objects will be precisely circles and rings, as can be seen from Figure 3. The resulting image can be enlarged, however, if the images of two neighboring point objects merge, they will continue to merge. This kind of magnification is often said to be useless - larger images will simply be blurrier. An example of useless magnification is shown in Figure 4. The formula is often called the diffraction limit, and it is so famous that it was carved on the monument to the author of this formula, the German optical physicist Ernst Abbe.
Of course, over time, optical microscopes began to be equipped with a variety of devices that made it possible to store images. The human eye was first supplemented by film cameras and films, and then by cameras based on digital devices that convert the light falling on them into electrical signals. The most common of these devices are CCD matrices (CCD stands for charge-coupled device). The number of pixels in digital cameras continues to increase, but this alone cannot improve the resolution of optical microscopes.
Even twenty-five years ago it seemed that the diffraction limit was insurmountable and that in order to study objects whose dimensions are many times smaller than the wavelength of light, it was necessary to abandon light as such. This is exactly the path that the creators of electron and X-ray microscopes took. Despite the numerous advantages of such microscopes, the problem of using light to view nanoobjects remained. There were many reasons for this: convenience and ease of working with objects, the short time required to obtain an image, known methods for coloring samples, and much more. Finally, after years of hard work, it became possible to view nanoscale objects using an optical microscope. The greatest progress in this direction has been achieved in the field of fluorescence microscopy. Of course, no one has canceled the diffraction limit, but they managed to get around it. Currently, there are various optical microscopes that make it possible to examine objects whose dimensions are much smaller than the wavelength of the very light that creates images of these objects. All these devices share one common principle. Let's try to explain which one it is.
From what has already been said about the diffraction limit of resolution, it is clear that seeing a point source is not that difficult. If this source is of sufficient intensity, its image will be clearly visible. The shape and size of this image, as already mentioned, will be determined by the properties of the optical system. At the same time, knowing the properties of the optical system and being sure that the object is a point object, you can determine exactly where the object is located. The accuracy of determining the coordinates of such an object is quite high. This can be illustrated by Figure 5. The coordinates of a point object can be determined more accurately, the more intensely it glows. Back in the 80s of the last century, using an optical microscope, they were able to determine the position of individual luminous molecules with an accuracy of 10–20 nanometers. A necessary condition for such an accurate determination of the coordinates of a point source is its loneliness. The closest other point source must be so far away that the researcher knows for sure that the image being processed corresponds to one source. It is clear that this is a distance l must satisfy the condition. In this case, image analysis can provide very precise data on the position of the source itself.
Most objects whose dimensions are much smaller than the resolution of an optical microscope can be represented as a set of point sources. The light sources in such a set are located from each other at distances much smaller than . If these sources shine simultaneously, then it will be impossible to say anything about where exactly they are located. However, if you can make these sources shine in turn, then the position of each of them can be determined with high accuracy. If this accuracy exceeds the distance between the sources, then, having knowledge of the position of each of them, one can find out what their relative positions are. This means that information has been obtained about the shape and size of the object, which is presented as a set of point sources. In other words, in this case, you can examine an object with an optical microscope whose dimensions are smaller than the diffraction limit!
Thus, the key point is to obtain information about different parts of a nanoobject independently of each other. There are three main groups of methods to do this.
The first group of methods purposefully makes one or another part of the object under study shine. The best known of these methods is near-field scanning optical microscopy. Let's take a closer look at it.
If you carefully study the conditions that are implied when talking about the diffraction limit, you will find that the distances from objects to lenses are much greater than the wavelength of light. At distances comparable to and smaller than this wavelength, the picture is different. Near any object caught in the electromagnetic field of a light wave, there is an alternating electromagnetic field, the frequency of change of which is the same as the frequency of change of the field in the light wave. Unlike a light wave, this field quickly decays as it moves away from the nanoobject. The distance at which the intensity decreases, e.g. e times, comparable to the size of the object. Thus, the electromagnetic field of optical frequency turns out to be concentrated in a volume of space, the size of which is much smaller than the wavelength of light. Any nanoobject that falls into this area will interact in one way or another with the concentrated field. If the object with the help of which this field concentration is carried out is sequentially moved along any trajectory along the nanoobject being studied and the light emitted by this system is recorded, then an image can be constructed from individual points lying on this trajectory. Of course, at each point the image will look as shown in Figure 2, but the resolution will be determined by how much the field was concentrated. And this, in turn, is determined by the size of the object with the help of which this field is concentrated.
The most common way to concentrate the field this way is to make a very small hole in a metal screen. Typically, this hole is located at the end of a pointed light guide coated with a thin film of metal (light guide is often called optical fiber and is widely used for transmitting data over long distances). Now it is possible to produce holes with diameters from 30 to 100 nm. The resolution is the same in size. Devices operating on this principle are called near-field scanning optical microscopes. They appeared 25 years ago.
The essence of the second group of methods comes down to the following. Instead of making nearby nanoobjects shine in turn, you can use objects that glow in different colors. In this case, with the help of light filters that transmit light of one color or another, you can determine the position of each object, and then create a single picture. This is very similar to what is shown in Figure 5, only the colors will be different for the three images.
The last group of methods that make it possible to overcome the diffraction limit and examine nanoobjects uses the properties of the luminous objects themselves. There are sources that can be “turned on” and “turned off” using specially selected light. Such switchings occur statistically. In other words, if there are many switchable nanoobjects, then by selecting the wavelength of light and its intensity, you can force only part of these objects to “turn off”. The remaining objects will continue to shine, and an image can be obtained from them. After this, you need to “turn on” all the sources and “turn off” some of them again. The set of sources that remain “on” will be different from the set that remained “on” the first time. By repeating this procedure many times, you can get a large set of images that differ from each other. By analyzing such a set, it is possible to locate a large proportion of all sources with very high accuracy, well above the diffraction limit. An example of super-resolution obtained in this way is shown in Figure 6.
Super-resolution optical microscopy is currently developing rapidly. It is safe to assume that this area will attract an increasing number of researchers in the coming years, and we hope that the readers of this article will be among them.
Guidelines
To study objects that are small in size and indistinguishable to the naked eye, special optical instruments are used - microscopes. Depending on the purpose, they are distinguished: simplified, working, research and universal. According to the illumination source used, microscopes are divided into: light, fluorescent, ultraviolet, electronic, neutron, scanning, tunnel. The design of any of the listed microscopes includes mechanical and optical parts. The mechanical part serves to create observation conditions - placing the object, focusing the image, the optical part - obtaining an enlarged image.
Light microscope device
A microscope is called a light microscope because it provides the ability to study an object in transmitted light in a bright field of view. (Fig. External view of Biomed 2) shows a general view of the Biomed-2 microscope.
|
|
The mechanical part of the microscope consists of a microscope base, a movable stage and a revolving device.
Focusing on an object is accomplished by moving the stage by rotating the coarse and fine adjustment knobs.
The coarse focusing range of the microscope is 40 mm.
The condenser is mounted on a bracket and located between the object stage and the collector lens. Its movement is made by rotating the condenser height adjustment knob. Its general view is shown in (Fig.???) A two-lens condenser with an aperture of 1.25 provides illumination of the fields on the object when working with lenses with magnification from 4 to 100 times.
The object table is mounted on a bracket. Coordinate movement of the object table is possible by rotating the handles. The object is secured to the table using drug holders. The holders can be moved relative to each other.
The coordinates of the object and the amount of movement are measured on scales with a division value of 1 mm and verniers with a division value of 0.1 mm. The range of object movement in the longitudinal direction is 60 mm, in the transverse direction – 40 mm. Condenser
Condenser
The microscope is equipped with a condenser mounting unit with the possibility of centering and focusing movement.
The basic microscope uses a universal condenser installed in a holder; when using immersion oil, the numerical aperture is 1.25.
When adjusting the lighting, a smooth change in the numerical aperture of the beam of rays illuminating the drug is carried out using the aperture diaphragm.
The condenser is installed in the condenser holder in a fixed position and secured with a locking screw.
Condenser centering screws are used during the illumination adjustment process to move the condenser in a plane perpendicular to the optical axis of the microscope while centering the field diaphragm image relative to the edges of the field of view.
The condenser up and down handle, located on the left side of the condenser holder bracket, is used when adjusting the lighting to focus on the image of the field diaphragm.
The filters are installed in a rotating ring located at the bottom of the condenser.
Optical part of the microscope
Consists of lighting and observation systems. The lighting system evenly illuminates the field of view. The observation system is designed to enlarge the image of the observed object.
Lighting system
It is located under the object table. It consists of a collector lens installed in the body, which is screwed into the hole in the base of the microscope and a socket with a lamp installed in it. The lamp socket is installed inside the base of the microscope. The microscope illuminator is powered from an alternating current network through a three-pin power cord connected to the power supply using a plug. The illuminator lamp is turned on by a switch located on the base of the microscope.
Observation system
Consists of lenses, monocular attachment and eyepieces.
Lenses
The lenses make up the most important, most valuable and fragile part of the microscope. Magnification, resolution and image quality depend on them. They are a system of mutually centered lenses enclosed in a metal frame. At the upper end of the frame there is a thread with which the lens is mounted in the socket of the revolver. The front (closest to the object) lens in the lens is called the frontal lens, and is the only one in the lens that produces magnification. All other objective lenses are called correction lenses and serve to correct deficiencies in the optical image.
When a beam of light rays with different wavelengths passes through the lenses, a rainbow coloration of the image occurs - chromatic aberration. Uneven refraction of rays on the curved surface of the lens leads to spherical aberration, which occurs due to uneven refraction of the central and peripheral rays. As a result, the dot image appears as a blurry circle.
The lenses included in the microscope kit are designed for an optical tube length of 160 mm, a height of 45 mm and a cover glass thickness of mm.
Objectives with magnifications greater than 10X are equipped with spring-loaded frames that protect the specimen and front lenses from damage when focusing on the surface of the specimen.
A colored ring can be applied to the lens body in accordance with the magnification, as well as:
- numerical aperture;
- optical tube length 160;
- cover glass thickness 0.17, 0 or －";
- type of immersion - oil OIL (MI) or water VI;
Objectives marked 0.17 are designed for studying preparations only with cover glasses 0.17 mm thick. Objectives marked 0 are designed for studying preparations only without cover glasses. Low magnification objectives (2.5 - 10), as well as immersion objectives, can be used when examining preparations with or without a cover glass. These lenses are marked with a － icon.
Eyepieces
The microscope eyepiece consists of two lenses: an eye lens (upper) and a collecting lens (lower). Between the lenses is the diaphragm. The diaphragm blocks side rays and transmits those close to the optical axis, which enhances the contrast of the image. The purpose of the eyepiece is to magnify the image produced by the lens. The eyepieces have their own magnification of ×5, ×10, ×12.5, ×16 and ×20, which is indicated on the frame.
The choice of eyepieces depends on the set of lenses used. When working with achromat, achrostigmata and achrofluar lenses, it is advisable to use eyepieces with a linear field of view of no more than 20 mm, with planchromat and planapochromat lenses - eyepieces with a linear field of view of 20; 22 and 26.5 mm.
Additionally, the microscope can be equipped with a WF10/22 eyepiece with a scale; scale division value is 0.1 mm.
Characteristics of microscopes
Microscope Magnification
The main characteristics of a microscope include magnification and resolution. The total magnification provided by a microscope is defined as the product of the objective magnification and the eyepiece magnification. However, magnification does not indicate the quality of the image; it can be clear or unclear. The clarity of the resulting image is characterized by the resolution of the microscope, i.e. the smallest size of objects or their parts that can be seen using this device.
The total magnification Г of the microscope during visual observation is determined by the formula: Г = βok × βok, where:
βrev - lens magnification (marked on the lens); βok - eyepiece magnification (marked on the eyepiece).
The diameter of the field observed in the object, Add mm, is determined by the formula: Add = Add × βob. Doc – diameter of the ocular field of view (marked on the eyepiece) mm. The calculated values of microscope magnification and the diameter of the observed field at the object are given in Table 3.
| Lens magnification | Microscope magnification and observed field on an object with an eyepiece: |
|||||
|---|---|---|---|---|---|---|
| 5/26* | 10/22 | 15/16* | ||||
| G | Add, mm | G | Add, mm | G | Add, mm | |
| 4 | 20 | 4,0 | 50 | 4,5 | 64 | 3,75 |
| 10 | 50 | 2,0 | 100 | 1,8 | 160 | 1,5 |
| 20 | 100 | 1,0 | 200 | 0,9 | 320 | 0,75 |
| 40 | 200 | 0,5 | 420 | 0,45 | 640 | 0,38 |
| 100 | 500 | 0,2 | 1000 | 0,18 | 1600 | 0,15 |
- By additional order
Microscope resolution
The resolution of a microscope is determined by the minimum (resolving) distance between two points (or two thinnest lines) visible separately, and is calculated by the formula
D=λ/(A1+A2) , where d is the minimum (resolution) distance between two points (lines); λ is the wavelength of the light used; A1 and A2 are the numerical aperture of the lens (marked on its frame) and condenser.
You can increase the resolution (i.e. reduce the absolute value of d, since these are reciprocal values) in the following ways: illuminate the object with light with a shorter wavelength λ (for example, ultraviolet or short-wave rays), use lenses with a larger aperture A1, or increase the aperture condenser A2.
Lens working distance
Microscopes are equipped with four removable objectives with their own magnifications of 4×, 10×, 40× and 100×, marked on a metal frame. Lens magnification depends on the curvature of the main front lens: the greater the curvature, the shorter the focal length and the greater the magnification. This must be remembered when microscopying - the greater the magnification provided by the lens, the smaller the free working distance and the lower it should be lowered above the plane of the specimen.
Immersion
All lenses are divided into dry and immersion, or submersible. A lens is called dry if there is air between the front lens and the specimen in question. In this case, due to the difference in the refractive index of glass (1.52) and air (1.0), some of the light rays are deflected and do not enter the observer’s eye. Dry system lenses typically have a long focal length and provide low (10x) or medium (40x) magnification.
Immersion or submersible lenses are those lenses in which a liquid medium with a refractive index close to the refractive index of glass is placed between the front lens and the specimen. Cedar oil is usually used as an immersion medium. You can also use water, glycerin, transparent oils, monobromonaphthalene, etc. In this case, a homogeneous (homogeneous) medium is established between the front lens of the objective lens and the preparation (glass of the preparation - oil - lens glass) with the same refractive index. Thanks to this, all the rays, without refraction or changing direction, enter the lens, creating conditions for the best illumination of the drug. The value (n) of the refractive index is 1.33 for water, 1.515 for cedar oil, and 1.6 for monobromonaphthalene.
Microscopy technique
The microscope is connected to the electrical network using a power cable. Using a revolver, a lens with a magnification of ×10 is installed in the beam path. A slight stop and the clicking sound of the revolver spring indicate that the lens is mounted along the optical axis. Using the coarse focusing knob, lower the lens to a distance of 0.5 - 1.0 cm from the stage.
Rules for working with dry lenses.
The prepared preparation is placed on the stage and secured with a clamp. Using a ×10 dry lens, multiple fields of view are viewed. The stage is moved using side screws. The area of the drug required for examination is placed in the center of the field of view. Raise the tube and, by rotating the revolver, move the lens with a magnification of ×40, observing from the side, using a macrometric screw, lower the tube with the lens again almost until it comes into contact with the specimen. Look into the eyepiece and very slowly lift the tube until the contours of the image appear. Precise focusing is carried out using a micrometer screw, rotating it in one direction or another, but not more than one full turn. If resistance is felt when rotating the micrometer screw, it means that its stroke has been completed. In this case, turn the screw one or two full turns in the opposite direction, again find the image using the macrometric screw and proceed to work with the micrometric screw.
It is useful to train yourself to keep both eyes open when microscopying and to use them alternately, as this will fatigue your eyesight less.
When changing lenses, one should not forget that the resolution of the microscope depends on the ratio of the aperture of the lens and the condenser. The numerical aperture of the objective with a magnification of ×40 is 0.65, and that of the non-immersed condenser is 0.95. They can practically be brought into line by the following technique: having focused the specimen with the lens, remove the eyepiece and, looking through the tube, cover the iris diaphragm of the condenser until its edges become visible at the border of the uniformly illuminated rear lens of the lens. At this point, the numerical apertures of the condenser and objective will be approximately equal.
Rules for working with an immersion lens.
A small drop of immersion oil is applied to the preparation (preferably fixed and colored). The revolver is rotated and an immersion lens with a magnification of 100× is installed along the central optical axis. The condenser is lifted up until it stops. The iris diaphragm of the condenser is opened completely. Looking from the side, use a macrometric screw to lower the tube until the lens is immersed in oil, almost until the lens comes into contact with the slide of the specimen. This must be done very carefully so that the front lens does not move and become damaged. They look into the eyepiece, very slowly rotate the macrometric screw towards themselves and, without lifting the lens from the oil, lift the tube until the contours of the object appear. It should be remembered that the free working distance in the immersion lens is 0.1 - 0.15 mm. Then precise focusing is done using a macrometric screw. Several fields of view are examined in the preparation, moving the table with side screws. Upon completion of work with the immersion lens, lift the tube, remove the preparation and carefully wipe the front lens of the lens, first with a dry soft cotton napkin, then with the same napkin, but slightly moistened with pure gasoline. Oil should not be left on the surface of the lens, as it allows dust to settle and can lead to damage to the microscope optics over time. The preparation is freed from oil first with a piece of filter paper, then the glass is treated with gasoline or xylene.
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