The resolution of the microscope is called. Microscope as an optical instrument. Resolution of the microscope. Classification of light microscopes. Geometric system enlargement

2. The optical system of the microscope.

3. Magnification of the microscope.

4. Resolution limit. Resolution of the microscope.

5. Useful magnification of the microscope.

6. Special techniques of microscopy.

7. Basic concepts and formulas.

8. Tasks.

The ability of the eye to distinguish fine details of an object depends on the size of the image on the retina or on the angle of view. To increase the angle of view, special optical devices are used.

25.1. Magnifier

The simplest optical device for increasing the angle of view is a magnifying glass, which is a short-focus converging lens (f \u003d 1-10 cm).

The object in question is placed between the magnifier and its front focusin such a way that its virtual image is within the accommodation for the given eye. Usually planes of far or near accommodation are used. The latter case is preferable, since the eye does not get tired (the annular muscle is not tense).

Let's compare the angles of view at which the object is seen, viewed "unarmed" normalwith the eye and with a magnifying glass. The calculations will be performed for the case when a virtual image of an object is obtained at infinity (the far limit of accommodation).

When examining an object with the naked eye (Fig. 25.1, a), in order to obtain the maximum angle of view, the object must be placed at the best vision distance a 0. The angle of view at which the object is seen is β \u003d B / a 0 (B is the size of the object).

When examining an object with a magnifying glass (Fig. 25.1, b), it is placed in the front focal plane of the magnifying glass. In this case, the eye sees an imaginary image of an object B "located in an infinitely distant plane. The angle of view at which the image is visible is β" ≈ B / f.

Figure: 25.1.Viewing angles: and- with the naked eye; b- using a magnifying glass: f - focal length of the magnifying glass; N - eye nodal point

Magnifying glass- angle of view ratioβ", under which you can see the image of an object in a magnifying glass, to the angle of viewβ, under which the object is visible to the "naked" normal eye from the distance of the best vision:

Magnifier magnifications are different for the myopic and farsighted eyes, since they have different distances for the best vision.

Let us give, without derivation, the formula for the magnification given by a magnifying glass used by a myopic or far-sighted eye when forming an image in the plane of distant accommodation:

where and distance is the far limit of accommodation.

Formula (25.1) allows us to assume that by decreasing the focal length of the magnifying glass, one can achieve an arbitrarily large magnification. In principle, this is so. However, with decreasing the focal length of the magnifying glass and maintaining its size, such aberrations arise that negate the entire effect of magnification. Therefore, single-lens magnifiers usually have 5-7x magnification.

To reduce aberrations, complex magnifiers are made, consisting of two or three lenses. In this case, a magnification of 50 times can be achieved.

25.2. Microscope optical system

Greater magnification can be achieved by viewing with a magnifying glass the actual image of an object created by another lens or lens system. Such an optical device is implemented in a microscope. The magnifying glass in this case is called eyepiece,and another lens - lens.The path of the rays in the microscope is shown in Fig. 25.2.

Object B is positioned close to the front focus of the objective (F rev) so that its actual magnified image B "is between the eyepiece and its front focus.

Figure: 25.2.Beam path in a microscope.

this gives the eyepiece a virtual magnified image B ", which the eye examines.

By changing the distance between the object and the lens, the image B "is in the plane of the eye's distant accommodation" (in this case, the eye does not get tired). For a person with normal vision, B "is located in the focal plane of the eyepiece, and B" is obtained at infinity.

25.3. Microscope magnification

The main characteristic of the microscope is its angular increase.This concept is analogous to the angular magnification of a magnifying glass.

Microscope magnification- angle of view ratioβ", under which you can see the image of the object in eyepiece,to the angle of viewβ, under which the object is visible to the "naked" eye from the distance of the best vision (a 0):

25.4. Resolution limit. Microscope resolution

One might get the impression that by increasing the optical length of the tube, one can achieve an arbitrarily large magnification and, therefore, see the smallest details of the object.

However, taking into account the wave properties of light shows that the size of small details, distinguishable with a microscope, is subject to limitations associated with diffractionlight passing through the lens opening. Due to diffraction, the image of the illuminated point turns out not to be a point, but small light circle.If the details (points) of the object under consideration are located far enough, then the lens will give their images in the form of two separate circles and they can be distinguished (Fig. 25.3, a). The smallest distance between distinguishable points corresponds to the "touching" of the circles (Fig. 25.3, b). If the points are very close, then the corresponding “circles” overlap and are perceived as one object (Fig. 25.3, c).

Figure: 25.3.Resolution

The main characteristic showing the capabilities of the microscope in this regard is resolution limit.

Resolution limitmicroscope (Z) - the smallest distance between two points of an object at which they are distinguishable as separate objects (i.e. perceived in a microscope as two points).

The inverse of the resolution limit is called resolution.The lower the resolution limit, the higher the resolution.

The theoretical resolution limit of a microscope depends on the wavelength of the light used for illumination and on angular aperturelens.

Angular aperture(u) - the angle between the extreme rays of the light beam entering the objective lens from the object.

Let us indicate, without derivation, the formula for the resolution limit of a microscope in air:

where λ is the wavelength of the light that illuminates the object.

Modern microscopes have an angular aperture of 140 °. If you accept λ \u003d 0.555 μm, then we obtain the value of Z \u003d 0.3 μm for the resolution limit.

25.5. Useful microscope magnification

Let us find out how large the microscope magnification should be at a given resolution limit of its objective. Let's take into account that the eye has its own resolution limit due to the structure of the retina. In Lecture 24, we obtained the following estimate for eye resolution limit:Z GL \u003d 145-290 microns. In order for the eye to be able to distinguish the same points that the microscope separates, an increase is required

This increase is called useful increase.

Note that when using a microscope to photograph an object in formula (25.4), instead of Z GL, the film resolution limit Z PL should be used.

Useful microscope magnification- magnification, at which an object having a size equal to the resolution limit of the microscope has an image, the size of which is equal to the resolution limit of the eye.

Using the above estimate for the resolution limit of the microscope Z m ≈0.3 μm), we find: Г п ~ 500-1000.

It makes no sense to achieve a greater value for the magnification of the microscope, since no additional details can be seen anyway.

Useful microscope magnification - it is a clever combination of the resolution of both the microscope and the eye.

25.6. Special techniques of microscopy

Special microscopy techniques are used to increase the resolution (decrease the resolution limit) of the microscope.

1. Immersion.In some microscopes, to reduce resolution limitthe space between the lens and the object is filled with a special liquid - immersion.Such a microscope is called immersion.The immersion effect is to reduce the wavelength: λ = λ 0 / n, where λ 0 - the wavelength of light in vacuum, and n is the refractive index of the immersion. In this case, the microscope resolution limit is determined by the following formula (generalization of formula (25.3)):

Note that special objectives are created for immersion microscopes, since the focal length of the objective changes in a liquid medium.

2. UV microscopy.For decreasing resolution limituse shortwave ultraviolet radiation invisible to the eye. In ultraviolet microscopes, a micro-object is examined in UV rays (in this case, the lenses are made of quartz glass, and registration is carried out on photographic film or on a special luminescent screen).

3. Measuring the size of microscopic objects.Using a microscope, you can determine the size of the observed object. For this, an ocular micrometer is used. The simplest eyepiece micrometer is a round glass plate with a graduated scale. The micrometer is installed in the plane of the image obtained from the objective. When viewed through the eyepiece, the images of the object and the scales merge, you can count the distance along the scale corresponds to the measured value. Preliminarily determine the price of division of the eyepiece micrometer from a known object.

4. Microprojection and microphotography.With a microscope, you can not only observe an object through the eyepiece, but also photograph it or project it onto a screen. In this case, special eyepieces are used, which project the intermediate image A "B" onto a film or screen.

5. Ultramicroscopy.The microscope can detect particles that are outside of its resolution. This method uses oblique lighting, due to which the microparticles are visible as light points against a dark background, while the structure of the particles cannot be seen, it is only possible to establish the fact of their presence.

The theory shows that no matter how strong the microscope is, any object less than 3 microns in size will appear in it simply as one point, without any details. But this does not mean that such particles cannot be seen, tracked or counted.

To observe particles that are smaller than the resolution limit of the microscope, a device called ultramicroscope.The main part of the ultramicroscope is a strong illumination device; particles illuminated in this way are observed in an ordinary microscope. Ultramicroscopy is based on the fact that small particles suspended in a liquid or gas are made visible under strong lateral illumination (remember the dust particles visible in a sunbeam).

25.8. Basic concepts and formulas

End of the table

25.8. Tasks

1. A lens with a focal length of 0.8 cm is used as a microscope objective with an eyepiece focal length of 2 cm. The optical tube length is 18 cm. What is the magnification of a microscope?

2. Determine the resolution limit for dry and immersion (n \u003d 1.55) objectives with an angular aperture of u \u003d 140 о. Take the wavelength equal to 0.555 microns.

3. What is the resolution limit at wavelength λ \u003d 0.555 microns, if the numerical aperture is: A 1 \u003d 0.25, A 2 \u003d 0.65?

4. With what refractive index should the immersion liquid be taken in order to view a subcellular element with a diameter of 0.25 μm in a microscope when observed through an orange filter (wavelength 600 nm)? The microscope aperture angle is 70 °.

5. On the rim of the magnifier there is an inscription “x10” Determine the focal length of this magnifier.

6. Focal length of the microscope objective f 1 \u003d 0.3 cm, tube length Δ \u003d 15 cm, magnification D \u003d 2500. Find the focal length F 2 of the eyepiece. The best vision distance is a 0 \u003d 25 cm.

Light microscopy

Light microscopy provides a magnification of up to 2-3 thousand times, a color and moving image of a living object, the possibility of microcinema and long-term observation of the same object, an assessment of its dynamics and chemistry.

The main characteristics of any microscope are resolution and contrast. Resolution is the minimum distance at which two points are separately demonstrated by the microscope. The human eye has a resolution of 0.2 mm for best vision.

Image contrast is the difference between the brightness of the image and the background. If this difference is less than 3 - 4%, then it cannot be captured either with the eye or with a photographic plate; then the image will remain invisible, even if the microscope resolves its details. Contrast is influenced both by the properties of the object, which change the luminous flux compared to the background, and by the ability of the optics to pick up the resulting differences in the properties of the beam.

The capabilities of a light microscope are limited by the wave nature of light. The physical properties of light - color (wavelength), brightness (wave amplitude), phase, density and direction of wave propagation change depending on the properties of the object. These differences are used in modern microscopes to create contrast.

Microscope magnification is defined as the product of the objective magnification times 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 of the microscopes have magnifications of up to 2000. Even higher magnifications do not make sense, since this resolution does not improve. On the contrary, the image quality deteriorates.

Numerical aperture is used to express the resolution of an optical system or the aperture of a lens. Lens Aperture - The intensity of light per unit area of \u200b\u200bthe image is approximately equal to the square of NA. The NA value is about 0.95 for a good lens. The microscope is usually sized to have a total magnification of about 1000 NA. If a liquid (oil or, more rarely, distilled water) is introduced between the objective and the sample, you get an "immersion" objective with an NA value of up to 1.4, and with a corresponding improvement in resolution.

Light microscopy methods

Light microscopy methods (lighting and observation). Microscopy methods are selected (and provided constructively) depending on the nature and properties of the objects under study, since the latter, as noted above, affect the image contrast.

Brightfield method and its varieties

The bright field method in transmitted light is used in the study of transparent preparations with absorbing (light-absorbing) particles and parts included in them. This can be, for example, thin colored sections of animal and plant tissues, thin sections of minerals, etc. In the absence of a preparation, a beam of light from the condenser passing through the lens gives a uniformly illuminated field near the focal plane of the eyepiece. In the presence of an absorbing element in the preparation, the light incident on it is partially absorbed and partially scattered, which causes the appearance of the image. It is possible to use the method when observing non-absorbent objects, but only if they scatter the illuminating beam so strongly that a significant part of it does not fall into the lens.

The oblique lighting method is a variation on the previous method. The difference between them is that the light is directed to the object at a large angle to the direction of observation. Sometimes this helps to reveal the "relief" of the object due to the formation of shadows.

Reflected light brightfield is used to study opaque objects that reflect light, such as metal or ore sections. Illumination of the preparation (from an illuminator and a semitransparent mirror) is performed from above, through the lens, which simultaneously plays the role of a condenser. In the image created in the plane by the objective together with the tube lens, the structure of the preparation is visible due to the difference in the reflectivity of its elements; in the bright field, inhomogeneities are also distinguished, scattering the light incident on them.

Darkfield method and its varieties

Dark-field microscopy is used to produce images of transparent, non-absorbent objects that cannot be seen with the bright field method. These are often biological objects. The light from the illuminator and the mirror is directed to the preparation by a special condenser - the so-called. dark field condenser. Upon exiting the condenser, the main part of the light rays, which did not change their direction when passing through the transparent preparation, forms a beam in the form of a hollow cone and does not enter the lens (which is located inside this cone). The image in the microscope is formed with the help of only a small part of the rays scattered by microparticles of the preparation on the slide inside the cone and passed through the objective. Dark-field microscopy is based on the Tyndall effect, a well-known example of which is the detection of dust particles in air when illuminated with a narrow beam of sunlight. In the field of view, against a dark background, light images of the structural elements of the preparation, which differ from the environment in refractive index, are visible. For large particles, only light edges are visible, scattering light rays. Using this method, it is impossible to determine by the appearance of the image whether particles are transparent or opaque, they have a higher or lower refractive index compared to the environment.

Conducting dark field research

Slides should be no thicker than 1.1-1.2 mm, cover slides 0.17 mm, no scratches or dirt. When preparing the drug, the presence of bubbles and large particles should be avoided (these defects will be visible brightly glowing and will not allow observing the drug). For dark-field, more powerful illuminators and maximum lamp glow are used.

Darkfield lighting setup is basically as follows:

Set the light over Koehler;

Replace the bright-field condenser with a dark-field one;

Immersion oil or distilled water is applied to the upper lens of the condenser;

Raise the condenser until it touches the bottom surface of the slide;

Low magnification lens focuses on the specimen;

With the help of centering screws, a bright spot (sometimes having a darkened central area) is transferred to the center of the field of view;

Raising and lowering the condenser, they achieve the disappearance of the darkened central area and obtain an evenly illuminated bright spot.

If this is not possible, then it is necessary to check the thickness of the glass slide (usually this phenomenon is observed when using too thick glass slides - the cone of light is focused in the thickness of the glass).

After correct adjustment of the light, the objective of the required magnification is installed and the preparation is examined.

The method of ultramicroscopy is based on the same principle - preparations in ultramicroscopes are illuminated perpendicular to the direction of observation. With this method, it is possible to detect (but not "observe" in the literal sense of the word) extremely small particles, sizes of which lie far beyond the resolution of the most powerful microscopes. With the help of immersion ultramicroscopes, it is possible to register the presence of particles in a preparation with particles up to 2 × 10 in -9 degrees m. But the shape and exact dimensions of such particles cannot be determined using this method. Their images are presented to the observer in the form of diffraction spots, the sizes of which do not depend on the size and shape of the particles themselves, but on the objective aperture and the magnification of the microscope. Since these particles scatter very little light, they require extremely strong light sources, such as a carbon arc, to illuminate them. Ultramicroscopes are mainly used in colloidal chemistry.

Phase contrast method

The phase contrast method and its variety - the so-called. method of "anoptral" contrast are intended for obtaining images of transparent and colorless objects invisible when observed by the bright field method. These include, for example, live unpainted animal tissue. The essence of the method is that even with very small differences in the refractive indices of different elements of the preparation, the light wave passing through them undergoes different changes in phase (acquires the so-called phase relief). Not perceived directly by either the eye or the photographic plate, these phase changes with the help of a special optical device are converted into changes in the amplitude of the light wave, ie, into changes in brightness ("amplitude relief"), which are already distinguishable by the eye or are fixed on the photosensitive layer. In other words, in the resulting visible image, the distribution of brightness (amplitudes) reproduces the phase relief. The resulting image is called phase contrast.

The phase contrast device can be installed on any light microscope and consists of:

A set of objectives with special phase plates;

Swing disc condenser. It has annular diaphragms corresponding to the phase plates in each of the objectives;

Auxiliary telescope for adjusting phase contrast.

Phase contrast adjustment is as follows:

Replace the objectives and the microscope condenser with phase lenses (designated by the letters Ph);

Install a low magnification lens. The hole in the condenser disc must be without an annular diaphragm (indicated by the number "0");

Tune the light according to Koehler;

Choose a phase lens of appropriate magnification and focus it on the specimen;

Rotate the condenser disk and install the annular diaphragm corresponding to the lens;

Methodical instructions

To study objects that are small and indistinguishable with the naked eye, special optical instruments are used - microscopes. Depending on the purpose, they are distinguished: simplified, working, research and universal. According to the light source used, microscopes are divided into: light, luminescent, ultraviolet, electronic, neutron, scanning, tunnel. The design of any of the listed microscopes includes mechanical and optical parts. The mechanical part is used to create observation conditions - to place the object, focus the image, the optical part - to obtain 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. The general view of the Biomed-2 microscope is shown in (Fig. Appearance of Biomed 2).

The mechanical part of the microscope consists of a microscope base, a movable stage and a revolving device.

Focusing on the object is carried out 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 is positioned between the stage and the collector lens. Its movement is made by turning 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 subject table is mounted on a bracket. Coordinate movement of the stage, possibly by rotating the handles. The object is fastened to the table by the specimen 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 graduation of 1 mm and vernier with a graduation of 0.1 mm. The range of movement of the object in the longitudinal direction is 60 mm, in the transverse direction - 40 mm. Condenser

Condenser

The microscope is equipped with a condenser attachment unit with the possibility of centering and focusing movements.

The microscope uses a universal condenser installed in a holder as a base; when using immersion oil, the numerical aperture is 1.25.

When adjusting the illumination, a smooth change in the numerical aperture of the beam of rays illuminating the preparation is carried out using an aperture diaphragm.

The condenser is inserted into 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 image of the field diaphragm relative to the edges of the field of view.

The up-down condenser 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 light 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

Located under the stage. It consists of a collector lens installed in a housing, which is screwed into the opening of the microscope base and a cartridge with a lamp installed in it. The lamp holder is installed inside the microscope base. The microscope illuminator is powered from the AC mains through a three-pin power cable, which is connected with a plug to the mains. The illuminator lamp is switched on by a switch located at the base of the microscope.

Observing system

Consists of objectives, monocular attachment and eyepieces.

Lenses

Objectives are the most important, most valuable and fragile part of a microscope. Magnification, resolution and image quality depend on them. They are a system of mutually centered lenses enclosed in a metal frame. There is a thread on the upper end of the barrel, with which the lens is attached to the revolver socket. The front (closest to the object) lens in the lens is called the frontal lens, the only one in the lens that produces magnification. All other objective lenses are called correction lenses and are used to eliminate the imperfections of 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. Unequal refraction of rays on the curved surface of the lens leads to spherical aberration due to uneven refraction of the central and peripheral rays. As a result, the dot image appears as a blurred circle.

Objectives included in the microscope set are designed for optical tube length 160 mm, height 45 mm and cover glass thickness mm.

Objectives with a magnification of more than 10X are equipped with spring-loaded frames that protect the specimen and the front lenses of the objectives from damage when focusing on the surface of the specimen.

A colored ring can be applied to the lens barrel in accordance with the magnification, as well as:

• numerical aperture;
• optical length of the tube 160;
• cover glass thickness 0.17, 0 or - ";
• type of immersion - oil OIL (MI) or water VI;

Objectives marked with 0.17 are designed to study specimens with only 0.17 mm cover slips. Objectives marked 0 are designed for examining slides only without cover slips. Objectives of low magnification (2.5 - 10), as well as immersion objectives can be used in the study of preparations both with a cover slip and without a cover slip. These lenses are marked with -.

Eyepieces

The microscope eyepiece consists of two lenses: an eye (upper) and a collective (lower). There is a diaphragm between the lenses. The diaphragm delays the side beams and transmits those close to the optical axis, which enhances the image contrast. The purpose of the eyepiece is to magnify the image provided by the lens. The eyepieces have their own magnification of × 5, × 10, × 12.5, × 16 and × 20 as indicated on the rim.

The choice of eyepieces depends on the set of lenses used. When working with objectives with achromats, achrostigmata and achrofluars, it is advisable to use eyepieces with a linear field of view not exceeding 20 mm, with planachromats and planapochromats - eyepieces with a linear field of view 20; 22 and 26.5 mm.

Additionally, the microscope can be equipped with an eyepiece WF10 / 22 with a scale; scale division value 0.1 mm.

Microscopes characteristics

Microscope magnification

The main characteristics of a microscope are magnification and resolution. The total magnification that a microscope gives is defined as the product of the objective magnification times the eyepiece magnification. However, the magnification does not characterize the quality of the image; it can be clear and unclear. The clarity of the resulting image is characterized by the resolution of the microscope, i.e. the smallest size of objects or their details that can be seen with this device.

The total magnification of the microscope G during visual observation is determined by the formula: G \u003d βok × βok, where:

βob - lens magnification (marked on the lens); βok - eyepiece magnification (marked on the eyepiece).

The diameter of the field observed in the object, Dob mm, is determined by the formula: Dock \u003d Dock × βob. Dock - diameter of the ocular field of view (marked on the eyepiece) mm. The calculated magnification of the microscope and the diameter of the observed field on the object are shown in Table 3.

• By additional order

Microscope resolution

The resolution of a microscope is determined by the minimum (resolution) distance between two points (or two thinnest strokes), visible separately, and is calculated by the formula

D \u003d λ / (A1 + A2), where d is the minimum (permissive) distance between two points (strokes); λ is the wavelength of the used light; A1 and A2 are the numerical apertures of the objective (marked on its barrel) and the condenser.

You can increase the resolution (i.e., decrease 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 wavelengths), use lenses with a larger A1 aperture, or increase the aperture condenser A2.

Lens working distance

The microscopes were equipped with four detachable objectives with their own magnifications of 4 ×, 10 ×, 40 × and 100 ×, indicated on a metal mount. The 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 during microscopy - the greater the magnification of the objective, the shorter the free working distance and the lower it should be lowered over the plane of the specimen.

Immersion

All lenses are divided into dry and immersion, or submersible. Dry is a lens that has air between its front lens and the drug in question. In this case, due to the difference in the refractive index of glass (1.52) and air (1.0), part of the light rays is deflected and does not enter the eye of the observer. Dry system lenses are usually long focal lengths and give low (10 ×) or medium (40 ×) magnifications.

Immersion, or submersible, are called such objectives, between the front lens of which and the drug is placed a liquid medium with a refractive index close to the refractive index of glass. Cedar oil is usually used as an immersion medium. You can also use water, glycerin, transparent oils, monobromnaphthalene, etc. In this case, a homogeneous (homogeneous) medium (drug glass - oil - objective glass) with the same refractive index is established between the frontal objective lens and the preparation. Due to this, all rays, without refraction and without changing direction, fall into the lens, creating the 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.

Microscopic 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 course of the rays. A slight stop and the sound of the revolver spring clicking indicate that the lens is mounted on the optical axis. Using the coarse focusing knob, lower the lens at a distance of 0.5 - 1.0 cm from the stage.

Rules for working with dry lenses.

The prepared preparation is placed on a stage and secured with a clamp. Using a dry lens with a magnification of × 10, several fields of view are viewed. The stage is moved with the side screws. The area of \u200b\u200bthe preparation required for the study is set in the center of the visual field. Raise the tube and rotate the revolver to transfer the lens with a magnification of × 40, observing from the side, again lower the tube with the lens with the macrometric screw until it almost touches the preparation. Look through the eyepiece, very slowly raise 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 during the rotation of the micrometer screw, it means that its stroke has been passed to the end. 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 during microscopy and use them alternately, as this will result in less eye fatigue.

When changing lenses, remember that the resolution of the microscope depends on the ratio of the lens aperture to the condenser. The numerical aperture of a lens with a magnification of × 40 is 0.65, that of a non-immersed condenser is 0.95. It is practically possible to bring them into compliance with the following technique: after focusing the preparation with the objective, remove the eyepiece and, looking into the tube, cover the condenser iris diaphragm until its edges become visible at the border of the uniformly illuminated rear objective lens. At this point, the numerical apertures of the condenser and the lens 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 turned and an immersion objective with a magnification of 100 × is installed along the central optical axis. The condenser is lifted up to the stop. The iris diaphragm of the condenser is fully opened. Looking from the side, the tube is lowered with a macrometric screw until the objective is immersed in oil, almost until the lens touches the specimen slide. This must be done very carefully so that the front lens does not move or get damaged. They look through the eyepiece, very slowly rotate the macrometric screw towards themselves and, without removing the lens from the oil, raise 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 carried out with a macrometric screw. Several visual fields are examined in the preparation by moving the table with lateral screws. At the end of work with the immersion lens, raise the tube, remove the preparation and carefully wipe the front lens of the objective, first with a dry soft cotton cloth, then with the same cloth, but slightly moistened with pure gasoline. Do not leave oil on the surface of the lens, as it contributes to the deposition of dust and can lead to damage to the microscope optics over time. The drug is freed from oil first with a piece of filter paper, then the glass is treated with gasoline or xylene.

Objective... Familiarization with the microscope device and determination of its resolution.

Devices and accessories: Microscope, small hole metal plate, illuminating mirror, ruler with scale.

Introduction

A microscope consists of an objective and an eyepiece, which are complex lens systems. The path of the rays in the microscope is shown in Fig. 1, in which the objective and eyepiece are represented by single lenses.

The subject AB is placed a little further from the main focus of the lens F about ... The objective of the microscope gives a real, reverse and enlarged image of the object (AB in Fig. 1), which is formed behind the double focal length of the objective. The enlarged image is viewed by the eyepiece as a magnifying glass. The image of the object viewed through the eyepiece, imaginary, reverse and magnified.

The distance between the back focus of the lens and the front focus of the eyepiece is called system optical spacing or optical length of the tube microscope .

The magnification of the microscope can be determined by the magnification of the objective and eyepiece:

N \u003d N about  N about \u003d ───── (1)

f about  f ok

where N about and N about - the magnification of the objective and eyepiece, respectively; D is the best vision distance for a normal eye (~ 25 cm);  is the optical length of the microscope tube; f about and f oK - the main focal lengths of the lens and eyepiece.

When analyzing formula (1), we can conclude that any small objects can be viewed in microscopes with high magnification. However, the useful magnification given by the microscope is limited by diffraction phenomena that become noticeable when viewing objects that are comparable in size to the length of the light wave.

Resolution Limit microscope is the smallest distance between points, the image of which in the microscope is obtained separately.

According to Abbe's theory, the resolution limit of the microscope is determined by the expression:

d \u003d ───── (2)

where d is the linear size of the item in question;  is the 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 beam (Fig. 2).

AT the quantity A \u003d nsin is called numerical aperture 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 objective and the wavelength of light that illuminates the object under consideration.

If the object is in the air (n \u003d 1), then in the microscope it is possible to distinguish points of the object, the distance between which:

d \u003d ─────

For microscopic objects, the angle  is close to 90 degrees, then sin  1, from which it follows that in a microscope it is possible to examine objects at a distance of ~ 0.61. In the case of visual observations (the maximum sensitivity of the eye falls on the green region of the visible spectrum   550 nm) in the microscope, you can see objects located at a distance of ~ 300 nm.

As follows from expression (2), the resolution of the microscope can be increased by decreasing the wavelength of the light that illuminates the object. So, when photographing objects in ultraviolet light ( ~ 250-300 nm), the resolution of the microscope can be doubled.

Microscope as an optical instrument. Resolution of the microscope.

A microscope (from micro ... and Greek skopeo - I look) is an optical device for obtaining a highly magnified image of a very small object under study, invisible to the naked eye. With the help of a microscope, you can see the small details of the structure of the object, the dimensions of which lie outside the resolution of the eye.

The human eye is a natural optical system that is characterized by a certain resolution. The resolution of the optical system is the smallest distance between the elements of the observed object, at which these elements can still be distinguished from one another (by the elements of an object we mean points or lines).

If the object is distant at the so-called best-seeing distance, which is 250 mm, then for a normal human eye the minimum resolution is about 0.1 mm, and for many people it is about 0.2 mm. This roughly corresponds to the thickness of a human hair. The dimensions of objects, such as plant and animal cells, small crystals, details of the microstructure of metals and alloys, etc., are much less than 0.1 mm. Such objects are usually called micro-objects. Microscopes of various types are intended for observation and study of such objects. Using a microscope, the shape, size, structure and many other characteristics of micro-objects are determined. An optical microscope makes it possible to distinguish structures with a distance between elements up to 0.20 μm, i.e. the resolution of such a microscope is about 0.20 μm or 200 nm.

When talking about the resolution of a microscope, they mean, as well as the resolution of the human eye, a separate image of two closely spaced objects. However, you need to understand that resolution and magnification are not the same thing. For example, if using imaging systemsget from a light microscope photographs of two lines located at a distance of less than 0.20 microns (i.e. less than the resolution of the microscope), then, no matter how we enlarge the image, the lines will still merge into one. Those. we can get a large magnification, but we will not improve its resolution. The total magnification of the microscope is equal to the product of the linear magnification of the objective and the angular magnification of the eyepiece. Magnification values \u200b\u200bare engraved on the mounts of objectives and eyepieces. Consider a flat field microscope (not stereoscopic). These are biological microscopes, metallographic, polarizing. Usually, the objectives of such a microscope have magnifications from 4 to 100 times, and the eyepieces - from 5 to 16. Therefore, the total magnification of an optical microscope is in the range from 20 to 1600 times. Of course, it is technically possible to develop and use in a microscope objectives and eyepieces that will give a total magnification significantly exceeding 1600x (for example, there are eyepieces with a magnification of 20x, which, when paired with a 100x objective lens, will give a magnification of 2000x). However, this is usually not practical. High magnifications are not an end in themselves in optical microscopy. The purpose of the microscope is to distinguish between the smallest possible structural elements of the preparation, i.e. to maximize the resolution of the microscope. And it has a limit due to the wave properties of light. Thus, a distinction is made between useful and unhelpful microscope magnification. A useful increase is when it is possible to reveal new details of the structure of an object, and an unhelpful one is an increase in which, by increasing the object hundreds or more times, it is impossible to find new details of the structure of the object.

Let us dwell once again on the concept of resolution. The resolving power of optical devices (also called the resolving power) characterizes the ability of these devices to give separate images of two points of an object close to each other. The smallest linear or angular distance between two points, starting from which their images merge, is called the linear or angular resolution limit. The existence of a resolution limit affects the choice of magnifications that we obtain with a microscope. Magnifications up to 1250 times are called useful, because with them we distinguish all the elements of the object's structure. In this case, the resolution capabilities of the microscope are exhausted. This magnification is obtained using a 100x oil immersion objective and a 12.5x eyepiece (useful eyepiece magnification ranges from 7.5x to 12.5x). At magnifications over 1250x, no new details of the specimen structure are revealed. However, sometimes such magnifications are used - in microphotography, when projecting images onto a screen, and in some other cases.

When a substantially higher useful magnification is required, an electron microscope is used. This microscope has a significantly higher resolution than an optical microscope. An electron microscope is a device for observing and photographing multiply (up to 106 times) enlarged images of objects, in which instead of light beams, beams of electrons accelerated to high energies (30-100 keV or more) are used in a deep vacuum.

Classification of light microscopes and their fields of application

By the structure of the optical circuitdistinguish between direct (objectives, attachment and eyepieces are located above the object) and inverted (the object is above the optical system that forms the image) microscopes. Also distinguish flat field microscopes(giving a two-dimensional image) and stereoscopic microscopes (volumetric - three-dimensional image).

By lighting methodsseparate microscopes of transmitted light (the image is formed by light passing through the object) and reflected light (the image is formed by light reflected from the surface of the object).

Microscopes can be divided also by research methods:

Bright field (a darker object stands out against a light background);

Dark field (against a dark background, a light object or its edge structures stand out);

Phase contrast (a dark gray relief object is observed on a light gray background);

Luminescence (luminous objects or parts of an object stand out against a dark background);

Polarized light (there is a brightly colored image of an object in different colors or shades).

The following areas of application of light microscopes can be distinguished:

Biological microscopes for laboratory biological and medical research of transparent objects. Available in bright and darkfield modes, phase contrast, polarized and fluorescent light.

Stereoscopic microscope in laboratories and in various industries for obtaining enlarged images of objects during working operations. Work in reflected and transmitted light is possible. Brightfield and darkfield modes are available.

Metallographic microscope in scientific and industrial laboratories for the study of opaque objects. Work in reflected light. Available in bright and dark field modes, phase contrast, polarized light.

Polarizing microscope in scientific and research laboratories for specialized research in polarized light. Work in reflected and transmitted light is possible. Brightfield and darkfield modes are available.

Objectives and eyepieces for microscopes

Microscope lens - a micro lens is a complex optical system that forms an enlarged image of an object, and is the main and most critical part of the microscope. The micro lens creates an actual inverted image that is viewed through the eyepiece.

Lenses differ in optical performance and design:

By the degree of correction of chromatic aberration: achromats, apochromats, etc.

With the corrected curvature of the image: - planachromats, planapromats.

The length of the microscope tube is 160 mm for transmitted light, 190 mm for reflected light, infinity for transmitted and reflected light;

By immersion properties: dry systems (without immersion) and immersion systems.

Apochromatic lenses differ from achromats in the degree of chromatic aberration correction. Due to the more perfect elimination of image defects associated with chromatic aberration, the image quality obtained when observing colored objects (colored sections, microorganisms, etc.), especially at high magnifications, is significantly higher when using apochromats. Apochromats and high magnification achromats are used in conjunction with compensating eyepieces. Apochromats are usually engraved with APO (APO). In achromats and apochromats, especially of high magnification, the curvature of the image field remains uncorrected.