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This article will discuss the magnification of a microscope, the units of measurement of this quantity, and methods for visually determining the resolving power of the device. We will also talk about the standard parameters of this value and methods for calculating the increase for a specific type of work.

Most often, the main power parameters of a microscope are indicated on the lens body. Unscrew the lens and inspect it. You can see two numbers written as a fraction. The first is magnification, the second is numerical aperture.

The aperture characterizes the device's ability to collect light and produce a clear image. The lens may also indicate the length of the tube and the thickness of the cover glass required for the job.

All about microscope magnification

Magnification is measured in multiples (x). The relationship of the eyepiece-lens system completely determines its significance. The product of the magnification of the eyepiece and the objective tells us about the working magnification that a given microscope creates. The dependence of the total magnification on the lens magnification is obvious. Based on power, lenses are divided into the following groups:

Small (no more than 10x);

Medium (up to 50x);

Large (over 50x);

Extra large (more than 100x).

The maximum objective magnification value for an optical microscope is 2000x. The eyepiece value is usually 10x and rarely changes. But the lens magnification varies widely (from 4 to 100x and 2000x).

When choosing a microscope, you need to consider who will be using it and what maximum magnification may be needed. For example, 200x is enough for a preschooler; school and university microscopes have a magnification of 400-1000x. But the research device should give at least 1500-2000x. This value allows you to work with bacteria and small cellular structures.

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Device resolution

What determines the clarity and quality of the image produced by a microscope? This is affected by the resolution of the device. To calculate this quantity, you need to find the quotient of the light wavelength and two numerical apertures. Therefore, it is determined by the condenser and the microscope lens. We remind you that the numerical aperture value can be seen on the lens barrel. The higher it is, the better the resolution of the device.

The optical microscope has a resolution limit of 0.2 microns. This is the minimum distance to the image when all points of the object are distinguishable.

Useful microscope magnification

We talk about useful magnification when the researcher's eye fully uses the resolving power of the microscope. This is achieved by observing the object at the maximum permissible angle. The useful magnification depends only on the numerical aperture and the type of lens. When calculating it, the numerical aperture increases by 500-1000 times.

A dry lens (only air between the object and the lens) creates a useful magnification of 1000x, i.e. NA is 1.

An immersion lens (a layer of immersion medium between the object and the lens) creates a useful magnification of 1250x, i.e. the numerical aperture is 1.25.

A blurred or fuzzy image indicates that the usable magnification is greater or less than the above values. Increasing or decreasing the specified value significantly degrades the performance of the microscope.

In this article we talked about the main characteristics of an optical microscope and methods for calculating them. We hope this information will be useful when working with this complex device.

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System expansion– an important factor, which is based on the choice of one or another microscope, depending on the solution of the necessary problems. We are all accustomed to the fact that it is necessary to inspect semiconductor elements on an inspection microscope with a magnification of 1000x or more, we can study insects by working with a 50x stereo microscope, and we studied onion scales, stained with iodine or brilliant green, at school on a monocular microscope, when the concept of magnification was not yet familiar to us.

But how to interpret the concept of magnification when we have a digital or confocal microscope in front of us, and the lenses have values ​​of 2000x, 5000x? What does this mean, will 1000x magnification on an optical microscope produce an image similar to a 1000x digital microscope? You will learn about this in this article.

Optical zoom system

When we work with a laboratory or stereoscopic microscope, calculating the current magnification of the system is not difficult. It is necessary to multiply the magnification of all optical components of the system. Typically, in the case of a stereo microscope, this includes an objective lens, a zoom lens or magnifying drum, and eyepieces.
In the case of a conventional laboratory microscope, the situation is even simpler - the total magnification of the system = the magnification of the eyepieces multiplied by the magnification of the lens installed in the working position. It is important to remember that sometimes there are specific models of microscope tubes that have a magnification or reduction factor (especially common with older models of Leitz microscopes). Also, additional optical components, be it a coaxial illumination source in a stereo microscope or an intermediate camera adapter located under the tube, may have an additional magnification factor.

For example, a stereo microscope with 10x eyepieces, a 2x objective, a zoom lens at 8x and a coaxial illumination unit with a factor of 1.5x will have a total optical magnification of 10x2x8x1.5 = 240x.

In this case, optical magnification (G) should be understood as the ratio of the tangent of the angle of inclination of the beam emerging from the optical system into image space to the tangent of the angle of the beam conjugate to it in the space of objects. Or the ratio of the length of the image of the segment formed by the optical system, perpendicular to the axis of the optical system, to the length of the segment itself

Geometric system magnification

In the case when the system does not have eyepieces, and the enlarged image is formed by a camera on a monitor screen, for example, as on a microscope, one should move on to the term geometric magnification of the optical system.
The geometric magnification of a microscope is the ratio of the linear size of the image of an object on the monitor to the real size of the object being studied.
You can get the geometric magnification value by multiplying the following values: optical magnification of the lens, optical magnification of the camera adapter, ratio of the monitor diagonal to the diagonal of the camera matrix.
For example, when working on a laboratory microscope with a 50x objective, a 0.5x camera adapter, a 1/2.5” camera and displaying the image on a 14” laptop monitor, we will get a geometric system magnification = 50x0.5x(14/0.4) = 875x.
Although the optical magnification will be equal to 500x in the case of 10x eyepieces.

Digital microscopes, confocal profilometers, electron microscopes and other systems that form a digital image of an object on a monitor screen operate with the concept of geometric magnification. This concept should not be confused with optical zoom.

Microscope resolution

There is a widespread misconception that the resolution of a microscope and its magnification are tightly linked - the higher the magnification, the smaller objects we can see through it. This is not true. The most important factor always remains permission optical system. After all, enlarging an unresolved image will not give us new information about it.

The resolution of the microscope depends on the numerical value of the objective aperture, as well as on the wavelength of the illumination source. As you can see, there is no system increase parameter in this formula.

where λ is the average wavelength of the light source, NA is the numerical aperture of the lens, R is the resolution of the optical system.

When using an NA 0.95 objective on a laboratory microscope with a halogen source (average wavelength about 500 nm), we obtain a resolution of about 300 nm.

As can be seen from schematic diagram light microscope, eyepieces magnify the actual image of the object. If, for example, you increase the magnification factor of the eyepieces by 2 times (insert 20x eyepieces into the microscope), then the total magnification of the system will double, but the resolution will remain the same.

Important Note

Let's assume that we have two options for building a simple laboratory microscope. We will build the first one using a 40x NA 0.65 objective and 10x eyepieces. The second one will use a 20x NA 0.4 objective and 20x eyepieces.

The magnification of microscopes in both versions will be the same= 400x (simple multiplication of objective and eyepiece magnification). And here the resolution in the first version will be higher, than in the second, since the numerical aperture of the 40x lens is larger. In addition, do not forget about the field of view of the eyepieces; for 20x this parameter is 20-25% lower.

The resolution of a microscope is characterized by the reciprocal of the linear resolution limit. According to Abbe's diffraction theory, the linear resolution limit of a microscope, i.e., the minimum distance between points of an object that are imaged as separate, is determined by the formula

where is the linear resolution limit; the wavelength of the light in which the observation is made; A is the numerical aperture, or simply the aperture, of a microscope (microlens).

From formula (324) it follows that to increase the resolution of the microscope, it is necessary to reduce the wavelength of light and increase the numerical aperture of the microscope. The first possibility is realized by photographing the objects under study in ultraviolet radiation.

The aperture of a microscope is determined by the formula where The value of the aperture angle of modern high-quality microlenses has been brought almost to the limit.

Another possibility for increasing the aperture is the use of an immersion liquid placed between the object in question and the microlens. Water, cedar oil, monobromonaphthalene are used as such a liquid.

In order for the observer's eye to fully use the resolution of the microscope, determined by formula (324), it is necessary to have an appropriate apparent magnification. If two points of the front focal plane of the optical system are located at a linear distance from each other (Fig. 157), then

Rice. 157. Diagram for determining the useful magnification of a microscope

angular distance between these points in image space

The observer's eye will perceive these points as separate if the angular distance between them is not less than the angular resolution limit of the eye

From formulas (325), (324) and (317) it follows that the apparent magnification of the microscope

Using the last formula, you can determine the minimum apparent magnification at which the observer's eye will fully use the resolving power of the microscope. This increase is called useful. When using formula (326), it should be borne in mind that in many cases the diameter of the exit pupil of the microscope is This leads to an increase in the angular limit of resolution of the eye to If we take the average wavelength in the visible region of the spectrum, then at the angular limit of resolution of the eye according to (326) for useful we obtain the magnification of the microscope.

Increase microscope is defined as the product of the objective magnification and the eyepiece magnification. Typical research microscopes have an eyepiece magnification of 10, and an objective magnification of 10, 45 and 100. Accordingly, the magnification of such a microscope ranges from 100 to 1000. Some microscopes have a magnification of up to 2000. Even higher magnification does not make sense, since resolution does not improve. On the contrary, the image quality deteriorates.

Formula for microscope magnification

Image quality is determined microscope resolution, i.e. the minimum distance at which the optics of a microscope can separately distinguish two closely spaced points. resolution depends on the numerical aperture of the objective, condenser and the wavelength of light with which the specimen is illuminated. The numerical aperture (opening) depends on the angular aperture and refractive index of the medium located between the front lens of the objective and condenser and the specimen.

In addition to the resolution of the system, the numerical aperture characterizes the lens aperture: the light intensity per unit image area is approximately equal to the square of NA. The NA value is approximately 0.95 for a good lens. The microscope is usually sized so that its total magnification is about 1000 NA.

Resolution limit– smallest distance. Between two closely spaced points of an object, visible through a microscope (perceived as two points).

Aperture (Latin apertura - hole) in optics - a characteristic of an optical device that describes its ability to collect light and resist diffraction blurring of image details. Depending on the type of optical system, this characteristic can be a linear or angular dimension. As a rule, among the parts of an optical device, a so-called aperture diaphragm is specially distinguished, which most strongly limits the diameters of the light beams passing through the optical instrument. Often, the role of such an aperture diaphragm is played by the frame or, simply, the edges of one of the optical elements (lenses, mirrors, prisms).

Angular aperture - the angle between the outer rays of a conical light beam at the entrance (exit) of the optical system.

Numerical aperture - is equal to the product of the refractive index of the medium between the object and the lens and the sine of the aperture angle. It is this value that most fully determines both the aperture ratio and the resolution of the microscope lens. To increase the numerical aperture of objectives in microscopy, the space between the objective and the cover glass is filled with immersion liquid.

Corner Objective aperture is the maximum angle (AOB) at which rays passing through the specimen can enter the lens. Numerical aperture lens is equal to the product of the sine of half the angular aperture and the refractive index of the medium located between the glass slide and the front lens of the lens. N.A. = n sinα where, N.A. - numerical aperture; n is the refractive index of the medium between the specimen and the lens; sinα is the sine of angle α equal to half the angle AOB in the diagram.

Thus, the aperture of dry systems (between the front objective lens and the air preparation) cannot be more than 1 (usually no more than 0.95). The medium placed between the specimen and the objective is called immersion liquid or immersion, and an objective designed to work with immersion liquid is called immersion. Thanks to immersion with more high rate refraction than air, you can increase the numerical aperture of the lens and, therefore, the resolution.

Numerical aperture lenses are always engraved on their frames.

The resolution of the microscope also depends on the aperture of the condenser. If we consider the condenser aperture to be equal to the lens aperture, then the resolution formula has the form R=λ/2NA, where R is the resolution limit; λ - wavelength; N.A - numerical aperture. From this formula it is clear that when observed in visible light (green part of the spectrum - λ = 550 nm), the resolution (resolution limit) of the microscope cannot be > 0.2 µm

Immersion (from Latin immersio - immersion) - a liquid that fills the space between the object of observation and a special immersion lens(condenser and glass slide). Three types of immersion liquids are mainly used: oil immersion (MI/Oil), water immersion (WI/W) and glycerol immersion (GI/Glyc), with the latter mainly used in ultraviolet microscopy.

Immersion is used in cases where it is necessary to increase the resolution of the microscope or its use requires technological process microscopy. This happens:

1. increasing visibility by increasing the difference in the refractive index of the medium and the object;

2. increasing the depth of the viewed layer, which depends on the refractive index of the medium.

In addition, immersion liquid can reduce the amount of stray light by eliminating glare from the subject. This eliminates the inevitable loss of light when it enters the lens.

Refraction of light - a change in the direction of light rays in a medium with a spatially varying refractive index n. Usually the term “R.” With." used to describe the propagation of optical fiber. radiation in inhomogeneous media with smoothly varying n from point to point (the trajectories of light rays in such media are smoothly curved lines). A sharp change in the direction of rays at the interface between two homogeneous media with different n is usually called. refraction of light. Atm. In optics and spectacle optics, the term “refraction” is traditionally used. Since the atmosphere is a heterogeneous medium, due to R. s. there is a shift in the apparent position of the celestial bodies relative to the true one, which must be taken into account in astronomy. R.s. in the atmosphere should also be taken into account when geodetic. measurements. R.s. is the cause of mirages. The phenomenon of R. s. allows you to visualize optical inhomogeneities in solid, liquid and gaseous media.

Refractometer and I ( from lat. refractus - refracted and Greek. metreo - measure) is a method for studying substances based on determining the index (coefficient) of refraction (refraction) and some of its functions. Refractometry (refractometric method) is used to identify chemical compounds, quantitative and structural analysis, and determine the physical and chemical parameters of substances.

The refractive index n is the ratio of the speed of light in the surrounding media. For liquids and solids n is usually determined relative to air, and for gases - relative to vacuum. The values ​​of n depend on the wavelength l of light and temperature, which are indicated in subscript and superscript, respectively. Refractometry methods are divided into two large groups: objective and subjective. Despite the undeniable advantage of objective methods, each objective study, as a rule, ends with correction by subjective methods. Objective methods. There are two subgroups of objective refractometry methods:

1. Objective in relation to the patient and subjective in relation to the doctor. An example is skiascopy, objective data of which can be obtained through a subjective assessment by a doctor of the skiascopic reflex of the subject.2. Objective in relation to both the subject and the researcher, implemented using a refractometric machine.

Polarization of light- physical optical characteristics radiation, describing the transverse anisotropy of light waves, i.e. nonequivalence of decomposition. directions in a plane perpendicular to the light beam. Creatures significance for understanding P. s. had its manifestation in the effects light interference and, in particular, the fact that two light beams with mutually perpendicular planes of polarization do not directly interfere. P.S. found natural explanation in el.-magn. theory of light, developed in 1865-73 by J. C. Maxwell, later in quantum electrodynamics.

The term wave polarization was introduced by Malus in relation to transverse mechanical waves

For receiving polarized light and its detection, there are special physical devices, called polarizers in the first case, and analyzers in the second. They are usually constructed in the same way. There are several ways to obtain and analyze polarized light.

1. Polarization using Polaroids. Polaroids are celluloid films coated with a thin layer of nodquinine sulfate crystals. The use of polaroids is currently the most common method of polarizing light.

2. Polarization by reflection. If a natural beam of light falls on a black polished surface, the reflected beam is partially polarized. As a polarizer and analyzer, mirror or fairly well-polished ordinary window glass, blackened on one side with asphalt varnish, can be used. The degree of polarization is greater, the more correctly the angle of incidence is maintained. For glass, the angle of incidence is 57°.

3. Polarization through refraction. The light beam is polarized not only upon reflection, but also upon

refraction. In this case, a stack is used as a polarizer and analyzer

10-15 thin glass plates folded together, located at an angle of 57° to the light rays incident on them.

Prism Nicolas (abbr. Nicole) is a polarizing device, the operating principle of which is based on the effects of birefringence and total internal reflection. The Nicolas prism consists of two identical triangular prisms made of Iceland spar, glued together with a thin layer of Canada balsam. The prisms are machined so that the end is beveled at an angle of 68° relative to the direction of transmitted light, and the glued sides make a right angle with the ends. In this case, the optical axis of the crystal ( AB) is at an angle of 64° with the direction of light.

The prism's full polarization aperture is 29°. A feature of the prism is the change in the direction of the emerging beam when the prism rotates, due to the refraction of the beveled ends of the prism. The prism cannot be used for ultraviolet polarization, since Canada balsam absorbs ultraviolet. Light with arbitrary polarization, passing through the end of the prism, experiences birefringence, splitting into two rays - an ordinary one, having a horizontal plane of polarization ( A.O.) and extraordinary, with a vertical plane of polarization ( AE). After which the ordinary beam experiences total internal reflection on the bonding plane and exits through the side surface. The Extraordinary comes out unhindered through the opposite end of the prism.

Brewster's Law - a law of optics that expresses the relationship of the refractive index with the angle at which light reflected from the interface will be completely polarized in a plane perpendicular to the plane of incidence, and the refracted beam is partially polarized in the plane of incidence, and the polarization of the refracted beam reaches its greatest value. It is easy to establish that in this case the reflected and refracted rays are mutually perpendicular. The corresponding angle is called Brewster's angle.

This optical phenomenon is named after the Scottish physicist David Brewster, who discovered it in 1815.

Brewster's Law : , Where n 12 - refractive index of the second medium relative to the first, θ Br- angle of incidence (Brewster angle).

When reflected from one plate at the Brewster angle, the intensity of linearly polarized light is very low (about 4% of the intensity of the incident beam). Therefore, in order to increase the intensity of the reflected light (or to polarize the light transmitted into the glass in a plane parallel to the plane of incidence), several bonded plates are used, folded into a stack - Stoletov’s foot. It is easy to trace what is happening in the drawing. Let a ray of light fall on the top of your foot. A fully polarized beam will be reflected from the first plate (about 4% of the original intensity), a fully polarized beam will also be reflected from the second plate (about 3.75% of the original intensity), and so on. In this case, the beam emerging from the bottom of the stack will become increasingly polarized in a plane parallel to the plane of incidence as plates are added. Concept complete refraction It has important for radio communications: most whip antennas radiate vertically polarized waves. Thus, if a wave hits the interface (ground, water or ionosphere) at the Brewster angle, there will be no reflected wave, and accordingly there will be no channel.

Malus's law - dependence of the intensity of linearly polarized light after its passage through the polarizer on the angle between the polarization planes of the incident light and the polarizer, where I 0 - intensity of light incident on the polarizer, I- intensity of light emerging from the polarizer. Light with a different (non-linear) polarization can be represented as the sum of two linearly polarized components, to each of which Malus’s law applies. According to Malus's law, the intensities of transmitted light are calculated in all polarization devices, for example, in polarization photometers and spectrophotometers. Reflection losses, depending on and not taken into account by the Malus law, are determined additionally.

Optically active substances , environments with natural optical activity. O.-a. V. are divided into 2 types. Those belonging to the 1st of them are optically active in any state of aggregation (sugars, camphor, tartaric acid), those belonging to the 2nd are active only in the crystalline phase (quartz, cinnabar). In substances of the 1st type, optical activity is due to the asymmetric structure of their molecules, of the 2nd type - by the specific orientation of molecules (ions) in the elementary cells of the crystal (asymmetry of the field of forces connecting particles in the crystal lattice). Crystals of O.-a. V. always exist in two forms - right and left; in this case, the lattice of the right crystal is mirror-symmetrical to the lattice of the left one and cannot be spatially combined with it (so-called enantiomorphic forms, see Enantiomorphism). Optical activity of the right and left forms of O.-a. V. Type 2 have different signs (and are equal in absolute value under the same external conditions), therefore they are called optical antipodes (sometimes O.-a.v. crystals of type 1 are also called this way ).

Rotation of the plane of polarization light - united by a common phenomenological manifestation of a group of effects consisting of rotation plane of polarization transverse wave as a result of interaction with an anisotropic medium. Naib. The effects associated with V.p.p. are well known. light, although similar phenomena are observed in other regions of the electromagnetic spectrum. waves (in particular, in the microwave range), as well as in acoustics, particle physics, etc.V. p.p. is usually due to the difference in coefficients. refraction of the medium for two circularly polarized (in the right and left circle) waves (the so-called circular anisotropy) and is described in the general case by an axial tensor of the second rank, connecting the axial vector of the rotation angle of the polarization plane with the polar wave vector. In a medium that has only circular anisotropy, a linearly polarized wave can be decomposed into two normal circularly polarized waves of equal amplitude (see. Normal fluctuations), the phase difference between them determines the azimuth of the plane of polarization of the total wave. In homogeneous media with circular anisotropy, the angle of the polarization linearly depends on the length of the path in the medium. Circular anisotropy can be either natural (spontaneous, inherent in the environment in an undisturbed state) or artificial, induced by external factors. influence. In the second case, circular asymmetry can be caused by the asymmetry of the disturbing influence or the combined symmetry properties of the medium and disturbance

Angle of rotation. The light beam can be natural and polarized. In a natural beam of light, vector oscillations occur in a disorderly manner.

Polarized light rays, in turn, are divided into linearly polarized ones, when vibrations occur in a straight line perpendicular to the beam; circularly polarized, when the end of the vector describes a circle in a plane perpendicular to the direction of the beam, and elliptically polarized, in which the oscillations occur along an ellipse.

The plane in which oscillations occur in a plane-polarized beam is called the plane of oscillation.

The plane passing through the direction of the polarized beam and perpendicular to the plane of oscillation is called the plane of polarization.

Light waves can be polarized using polarizer devices (Polaroid, tourmaline plate, Nicole, etc.).

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. General form it 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.

• 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. Multiple fields of view are viewed using a ×10 dry lens. 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 outline of the image appears. 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, find the image again using the macrometric screw and proceed to work with the micrometric screw.

It is useful to accustom 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 ×40 magnification is 0.65, and that of the non-immersed condenser is 0.95. It is practically possible to bring them into correspondence using 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. You should not leave oil 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.