The structure and main parts of an optical microscope. Microscope as an optical system Purpose of an optical microscope

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 reveal 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 of 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 is 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 some 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.

Lecture No. 7

Surface rendering methods

Optical microscopy

The human eye, which allows us to see and study the world around us, is a fairly simple optical system, the main element of which is the lens, which is actually a lens made of a liquid crystalline substance. The smallest objects that can be seen using such an optical system are about 0.1 mm in size, and to look at and study smaller objects, glasses or magnifying glasses were first used, and then complex structures made of optical lenses, called optical microscopes.

Microscope (from the Greek mikros - small and skopeo - I look) - a device for obtaining highly magnified images of objects (or details of their structure) invisible to the naked eye.

Optical design and operating principle of an optical microscope . One of the typical diagrams of an optical microscope is shown in Fig. 1. An object 7 located on a stage 10 is usually illuminated with artificial light from an illuminator (lamp 1 and a collector lens 2) using a mirror 4 and a condenser 6. To enlarge the object, a lens 8 and an eyepiece 9 are used. The lens creates a real inverted and enlarged 7" image of object 7. The eyepiece forms a secondary magnified virtual image of 7" usually at the best viewing distance D=250 mm. If the eyepiece is moved so that the 7" image is in front of the front focus of the eyepiece F approx, then the image given by the eyepiece becomes real and can be obtained on the screen or film. The total magnification is equal to the product of the lens magnification by the eyepiece magnification: x = bX approx. Magnification lens is expressed by the formula: b=D/F ob, where D is the distance between the rear focus of the lens F ob and the front focus of the eyepiece F approx (the so-called optical length of the microscope tube); F ob is the focal length of the lens. The magnification of the eyepiece is similar to the magnification of a magnifying glass. is expressed by the formula: X ok = 250/F ok, where F ok is the focal length of the eyepiece. Typically, the lenses of optical microscopes have magnifications from 6.3 to 100, and eyepieces from 7 to 15. Therefore, the total magnification of such a microscope ranges from 44 to 100. 1500. Field diaphragm 3 and aperture 5 serve to limit the light beam and reduce scattered light. An important characteristic of an optical microscope is its resolution, defined as the reciprocal of the smallest distance at which two adjacent structural elements can still be seen separately. The resolution of an optical microscope is limited due to light diffraction. Due to diffraction, the image of an infinitesimal luminous point, given by the lens of such a microscope, does not look like a point, but a round light disk (surrounded by dark and light rings), the diameter of which is equal to: d = 1.22 /A, Where  – wavelength of light and A–numerical aperture of the lens, equal to: A =nsin(a/2)(n– refractive index of the medium located between the object and the lens, a- the angle between the outer rays of a conical light beam emerging from a point on an object and entering the lens). If two luminous points are located close to each other, their diffraction patterns are superimposed on one another, giving a complex distribution of illumination in the image plane. The smallest relative difference in illumination that can be seen by the eye is 4%. This corresponds to the smallest distance resolved in an optical microscope, d=0.51 /A. For non-self-luminous objects, the maximum resolution is d etc amounts to  /(A+A"), Where A"– numerical aperture of the microscope condenser. Thus, the resolution ( 1/d) is directly proportional to the lens aperture and to increase it, the space between the object and the lens is filled with a liquid with a high refractive index. The apertures of high magnification immersion objectives reach A=1.3 (for conventional “dry” lenses A=0.9). The existence of a resolution limit influences the choice of optical microscope magnification. Optical microscope magnification within 500 A – 1000A is called useful, since with it the eye distinguishes all the elements of the structure of the object that are resolved by the microscope. At magnifications over 1000 A no new details of the object’s structure are revealed; Nevertheless, sometimes such magnifications are used, for example, in microphotography and microprojection.

Observation methods for optical microscopy . The structure of an object can be distinguished if different parts of it absorb and reflect light differently, or have different refractive indices from one another (or from the medium). These properties determine the difference in amplitudes and phases of light waves reflected or transmitted through different parts of the object, which, in turn, determines the contrast of the image. Therefore, observation methods used in optical microscopy are selected depending on the nature and properties of the object being studied.

Transmitted light field method used when studying transparent objects with absorbing (light-absorbing) particles and parts included in them. These are, for example, thin colored sections of animal and plant tissues, thin sections of minerals and radio electronics materials. In the absence of an object, a beam of rays from condenser 6 (see Fig. 1) passes through lens 8 and produces a uniformly illuminated field near the focal plane of eyepiece 9. If object 7 contains an absorbing object, then it partly absorbs and partly scatters the light incident on it ( dashed line), which, according to the diffraction theory, determines the appearance of the image. The method can also be useful for non-absorbing objects if they scatter the illuminating beam so strongly that a significant part of the beam does not reach the lens.

Bright field method in reflected light(Fig. 2) is used to observe opaque objects, for example, metal sections 4.

The object is illuminated from the illuminator 1 and the translucent mirror 2 from above through the lens 3, which simultaneously serves as a condenser. The image is created in plane 6 by the lens together with the tube lens 5; the structure of an object is visible due to differences in the reflectivity of its elements; In a bright field, inhomogeneities stand out, scattering the light incident on them.

Transmitted light dark field method(Fig. 3) is used to obtain images of transparent, non-absorbing objects. Light from illuminator 1 and mirror 2 passes through a special dark-field condenser 3 in the form of a hollow cone and does not directly enter the lens 5. The image is created only by light scattered by microparticles of the object 4. In the field of view 6, against a dark background, light images of particles that differ from the environment in refractive index are visible.

Ultramicroscopy method, based on the same principle (the illumination of an object in ultramicroscopes is perpendicular to the direction of observation), makes it possible to detect ultra-fine details, the dimensions of which (2 nm) lie far beyond the resolution of an optical microscope. The ability to detect such objects, for example, the smallest colloidal particles, using an ultramicroscope is due to the diffraction of light by them. Under strong lateral illumination, each particle in the ultramicroscope is marked by the observer as a bright point (luminous diffraction spot) on a dark background. Due to diffraction, very little light is scattered by the smallest particles. Therefore, in ultramicroscopy, strong light sources are usually used. Depending on the illumination intensity, light wavelength, and the difference between the refractive indices of the particle and the medium, the detected particles have sizes of (2–50) nm. It is impossible to determine the true size, shape and structure of particles from diffraction spots: an ultramicroscope does not provide images of optical objects under study. However, using an ultramicroscope, it is possible to determine the presence and numerical concentration of particles, study their movement, and also calculate the average size of particles if their weight concentration and density are known. The ultramicroscope was created in 1903. German physicist G. Siedentopf and Austrian chemist R. Zsigmondy. In the scheme of the slit ultramicroscope they proposed (Fig. 4, a) the system under study is motionless. Cuvette 5 with the object under study is illuminated by light source 1 (2 – condenser; 4 – lighting lens) through a narrow rectangular slit 3, the image of which is projected into the observation zone.

Through the eyepiece of observation microscope 6, luminous points of particles located in the image plane of the slit are visible. Above and below the illuminated area, the presence of particles is not detected. In a flow ultramicroscope (Fig. 4, b), the particles under study move along the tube towards the observer’s eye. As they cross the illumination zone, they are detected as bright flashes visually or using a photometric device. By adjusting the brightness of the illumination of the observed particles with a movable photometric wedge 7, it is possible to select for registration particles whose size exceeds a specified limit. Ultramicroscopes are used in studies of dispersed systems, to monitor the purity of atmospheric air, water, and the degree of contamination of optically transparent media with foreign inclusions.

When observing dark field method in reflected light(Fig. 5) opaque objects (for example, metal sections) are illuminated from above with a special ring system located around the lens and called epicondenser.

Rays of light from the dark field illuminator lamp 1, reflected from the epicondenser 2 and incident at an angle to the surface of the substrate 3, are scattered by foreign particles. These light rays scattered from foreign particles pass through the microscope objective lenses 4 and 5, are reflected from the microscope prism mirror 6 and, passing through the microscope eyepiece lens 7, are distinguished by the observer in the form of luminous points in a dark field.

Polarized light observation method(transmitted and reflected) is used to study anisotropic objects, such as minerals, ores, grains in thin sections of alloys, some animal and plant tissues and cells. Optical anisotropy is the difference in the optical properties of a medium depending on the direction of propagation of optical radiation (light) in it and its polarization. Light polarization is a physical characteristic of optical radiation that describes the transverse anisotropy of light waves, that is, the non-equivalence of different directions in a plane perpendicular to the light beam. The transverse nature of electromagnetic waves deprives the wave of axial symmetry relative to the direction of propagation due to the presence of selected directions (vectors E– electric field and vector strength N– magnetic field strength) in a plane perpendicular to the direction of propagation. Since the vectors E And N electromagnetic waves are perpendicular to each other, to fully describe the polarization state of a light beam, knowledge of the behavior of only one of them is required. Usually, vector E is chosen for this purpose. The light emitted by any individual elementary emitter (atom, molecule) is always polarized in each act of radiation. But macroscopic light sources consist of a huge number of such emitter particles; spatial orientations of vectors E and the moments of acts of light emission by individual particles are in most cases distributed chaotically. Therefore, in general radiation the direction E unpredictable at any given time. Such radiation is called unpolarized, or natural light. The light is called fully polarized, if two mutually perpendicular components (projections) of the vector E The light beam oscillates with a constant phase difference over time. Typically, the state of polarization of light is depicted using a polarization ellipse - a projection of the trajectory of the end of the vector E to a plane perpendicular to the beam (Fig. 6)

Optical anisotropy manifests itself in birefringence, changes in the polarization of light, and rotation of the polarization plane that occurs in optically active substances. The natural optical anisotropy of crystals is due to the dissimilarity in different directions of the field of forces connecting the atoms of the lattice. The natural optical activity of substances that exhibit it in any state of aggregation is associated with the asymmetry of the structure of individual molecules of such substances and the resulting difference in the interaction of these molecules with radiation of different polarizations, as well as with the characteristics of the excited states of electrons and “ionic cores” in optically active crystals . Induced (artificial) optical anisotropy occurs in media that are naturally optically isotropic under the influence of external fields that highlight a certain direction in such media. This can be an electric field, a magnetic field, a field of elastic forces, as well as a field of forces in a fluid flow. In the polarized light observation method, using analyzers and compensators that are included in the optical system, the change in the polarization of light passing through an object is studied.

Phase contrast method used to obtain images of transparent and colorless objects that are invisible when observed using the bright field method. Such objects include, for example, living, undyed animal tissue. The method is based on the fact that even with a small difference in the refractive indices of the object and the medium, the light wave passing through them undergoes different phase changes and acquires phase relief. These phase changes are converted into changes in brightness (“amplitude relief”) using a special phase plate (phase ring) located near the back focus of the lens. Rays passing through an object pass completely through the phase ring, which changes their phase by /4. At the same time, rays scattered in the object (deflected) do not fall into the phase ring and do not receive an additional phase shift. Taking into account the phase shift in the object, the phase difference between the rays deflected and non-deviated turns out to be close to 0 or /2, and as a result of the interference of light in the image plane of the object, they noticeably enhance or weaken each other, giving a contrast image of the structure of the object, in which the brightness distribution reproduces the above phase relief.

Interference contrast method consists in the fact that each beam entering the microscope bifurcates: one passes through the observed particle, and the second passes by it. In the eyepiece part of the microscope, both beams are again connected and interfere with each other. The result of interference is determined by the difference in the path of the rays d, which is expressed by the formula: d=N =(n 0 -n m )d 0 , where n 0 , n m are the refractive indices of the particle and the environment, respectively, d 0 – particle thickness, N– order of interference. A schematic diagram of one of the methods for implementing interference contrast is shown in Fig. 4. Condenser 1 and lens 4 are equipped with birefringent plates (marked in the figure with diagonal arrows), the first of which splits the original light beam into two beams, and the second reunites them. One of the rays, passing through object 3, is delayed in phase (acquires a path difference compared to the second ray); the magnitude of this delay is measured by compensator 5. The interference contrast method is in some respects similar to the phase contrast method - both of them are based on the interference of rays that have passed through a microparticle and passed it. The difference between the interference method and the phase contrast method lies mainly in the ability to measure with high accuracy (up to /300) the path differences introduced by a microobject using compensators. Based on these measurements, it is possible to make quantitative calculations, for example, of the total mass and concentration of dry matter in the cells of biological objects.

Research method in luminescence light is based on the fact that the green-orange glow of an object that occurs when it is illuminated with blue-violet or UV light is studied under a microscope. For this purpose, appropriate light filters are introduced before the condenser and after the microscope lens. The first of them transmits only radiation from the illuminator source that causes luminescence of the object, the second (after the lens) transmits only luminescence light to the observer’s eye. The method is used in microchemical analysis and flaw detection.

UV observation method allows you to increase the maximum resolution of the microscope, proportional to 1/. This method also expands the possibilities of microscopic studies due to the fact that particles of many substances, transparent in visible light, strongly absorb UV radiation of certain wavelengths and, therefore, are easily distinguishable in UV images. Images in UV microscopy are recorded either by photography or using an electron-optical converter or a luminescent screen.

IR observation method also requires converting an image invisible to the eye into a visible one by photographing it or using an electron-optical converter. IR microscopy allows you to study the internal structure of objects that are opaque in visible light, for example, dark glasses, some crystals, and minerals.

Main components of an optical microscope . In addition to the above optical components (for example, lens, eyepiece), an optical microscope also has a tripod or body, a stage for mounting the object under study, mechanisms for coarse and fine focusing, a device for mounting lenses and a tube for installing eyepieces. The use of one or another type of condenser (bright-field, dark-field, etc.) depends on the choice of the required observation method. The lenses in most modern optical microscopes are removable. Lenses vary:

a) according to spectral characteristics - for lenses for the visible region of the spectrum and for UV and IR microscopy (lens and mirror-lens);

b) along the length of the tube for which they are designed (depending on the design of the microscope);

c) according to the medium between the lens and the object - dry and immersion;

G ) according to the observation method - into conventional, phase-contrast, etc.

The type of eyepiece used for this observation method is determined by the choice of optical microscope lens. Adaptations for optical microscopes make it possible to improve observation conditions and expand research capabilities, carry out different types of illumination of objects, determine the size of objects, photograph objects through a microscope, etc. Types of microscopes are determined either by the area of ​​application or by the observation method. For example, biological microscopes designed for research in microbiology, histology, cytology, botany, medicine, as well as for observing transparent objects in physics, chemistry, etc. Metallographic microscopes designed for studying the microstructures of metals and alloys. Microphotographs of an unetched metal section taken using such a microscope are shown in Fig. 5 (a – in a bright field, b – with a phase-contrast device). Polarizing microscopes are equipped with additional polarizing devices and are intended mainly for studying thin sections of minerals and ores. Stereomicroscopes are used to obtain three-dimensional images of observed objects. Measuring microscopes Designed for various precision measurements in mechanical engineering. In addition to these groups of microscopes, there are specialized optical microscopes, for example: micro-installation for filming fast and slow processes (movement of microorganisms, processes of cell division, crystal growth, etc.); high temperature microscopes for studying objects heated to 2000°C; low magnification surgical microscopes, used during operations. Very complex instruments are microspectrophotometric installations for determining the absorption spectra of objects, television microimage analyzers, etc.

As already mentioned, regardless of the type of lenses used and the method of their connection, the resolution of optical microscopes is limited by the basic rule of optical technology, formulated back in 1873. (the so-called Rayleigh diffraction limit of resolution), according to which the minimum dimensions of the distinguishable details of the object under consideration cannot be less than half the wavelength of the light used for illumination. Since the shortest wavelengths in the range correspond to approximately 400 nm, the resolution of optical microscopes is fundamentally limited to half this value, that is, about 200 nm. The only way out of this situation was the creation of devices that use wave radiation with a shorter wavelength, that is, radiation of a non-light nature.

Electron microscopy

In quantum mechanics, an electron can be considered as a wave, which, in turn, can be influenced by electric or magnetic lenses (in complete analogy with the laws of conventional geometric optics). This is the basis of the operating principle of electron microscopes, which make it possible to significantly expand the possibilities of studying matter at the microscopic level (by increasing the resolution by orders of magnitude). In an electron microscope, instead of light, electrons themselves are used, which in this situation represent radiation with a much shorter wavelength (about 50,000 times shorter than light). In such devices, instead of glass lenses, electronic lenses (that is, fields of the appropriate configuration) are naturally used. Electron beams cannot propagate without scattering even in gaseous media, therefore a high vacuum (pressure up to 10–6 mmHg or 10–4 Pa) must be maintained inside the electron microscope along the entire electron path. Electron microscopes are divided into two large classes according to the method of application: transmission electron microscopes (TEM) and scanning (SEM) or, in other words, raster microscopes (SEM). The main difference between them is that in TEM an electron beam is passed through very thin layers of the substance under study, with a thickness of less than 1 micron (as if “transmitting” these layers through), and in scanning microscopes the electron beam is successively reflected from small areas of the surface ( the structure of the surface and its characteristic features can be determined by recording reflected electrons or secondary electrons arising during the interaction of the beam with the surface).

Transmission electron microscope (TEM) . The design of a TEM is similar to that of a conventional optical microscope (Fig. 1), only electrons (that is, their corresponding waves) are used instead of light rays. The first device of this type was created in 1932. German scientists M. Knoll and E. Ruska. In such a microscope, the light source is replaced by a so-called electron gun (electron source). The electron source is usually a heated tungsten or lanthanum hexaboride cathode. The cathode is electrically isolated from the rest of the device, and the electrons are accelerated by a strong electric field. The metal cathode 2 emits electrons, which are collected into a beam using a focusing electrode 3 and receive energy under the influence of a strong electric field in the space between the cathode and anode 1. To create this field, a high voltage of 100 kV or more is applied to the electrodes. The beam of electrons emerging from the electron gun is directed using a condenser lens 4 to the object in question, which scatters, reflects and absorbs electrons. They are focused by the objective lens 5, which creates an intermediate image of the object 7. The projection lens 6 again collects the electrons and creates a second, even more enlarged image of the object on the luminescent screen, on which, under the influence of the electrons, a luminous image of the object is created. Using a photographic plate placed under the screen, a photograph of the object in question is obtained.

Microscope(from Greek mikros- small and skopeo- I look) - an optical device for obtaining an enlarged image of small objects and their details invisible to the naked eye.

The first known microscope was created in 1590 in the Netherlands by hereditary opticians Zechariah And Hans Jansen , who mounted two convex lenses inside one tube. Later Descartes in his book “Dioptrics” (1637), he described a more complex microscope, composed of two lenses - a flat-concave (eyepiece) and a biconvex (objective). Further improvement of optics made it possible Anthony van Leeuwenhoek in 1674, made lenses with a magnification sufficient to carry out simple scientific observations and, for the first time in 1683, described microorganisms.

A modern microscope (Figure 1) consists of three main parts: optical, lighting and mechanical.

Main details optical part The microscope consists of two systems of magnifying lenses: an eyepiece facing the researcher’s eye and a lens facing the specimen. Eyepieces They have two lenses, the upper one is called the main one, and the lower one is called the collective lens. The eyepiece frames indicate what they produce. increase(×5, ×7, ×10, ×15). The number of eyepieces on a microscope may vary, and therefore monocular And binocular microscopes (designed to observe an object with one or two eyes), as well as trinoculars , allowing you to connect documentation systems (photo and video cameras) to the microscope.

Lenses are a system of lenses enclosed in a metal frame, of which the front (front) lens produces magnification, and the corrective lenses behind it eliminate defects in the optical image. The numbers on the lens frame also indicate what they produce. increase (×8, ×10, ×40, ×100). Most models intended for microbiological research are equipped with several lenses with different degrees of magnification and a rotating mechanism designed for quick change - turret , often called " turret ».

Lighting part is designed to create a light flux that allows you to illuminate an object in such a way that the optical part of the microscope performs its functions with extreme precision. The lighting part of a direct transmitted light microscope is located behind the object under the lens and includes Light source (lamp and electrical power supply) and optical-mechanical system (condenser, field and aperture adjustable diaphragm). Condenser consists of a system of lenses that are designed to collect rays coming from a light source at one point - focus , which must be in the plane of the object under consideration. In its turn d diaphragm located under the condenser and is designed to regulate (increase or decrease) the flow of rays passing from the light source.

Mechanical part The microscope contains parts that combine the optical and lighting parts described above, and also allow the placement and movement of the specimen under study. Accordingly, the mechanical part consists of grounds microscope and holder , to the top of which are attached tube - a hollow tube designed to accommodate the lens, as well as the above-mentioned turret. Below is stage , on which slides with the samples being studied are mounted. The stage can be moved horizontally using an appropriate device, as well as up and down, which allows for adjusting image sharpness using gross (macrometric) And precision (micrometric) screws.

Increase, which the microscope produces is determined by the product of the objective magnification and the eyepiece magnification. In addition to light-field microscopy, the following are widely used in special research methods: dark-field, phase-contrast, luminescent (fluorescent) and electron microscopy.

Primary(own) fluorescence occurs without special treatment of drugs and is inherent in a number of biologically active substances, such as aromatic amino acids, porphyrins, chlorophyll, vitamins A, B2, B1, some antibiotics (tetracycline) and chemotherapeutic substances (acriquin, rivanol). Secondary (induced) fluorescence occurs as a result of processing microscopic objects with fluorescent dyes - fluorochromes. Some of these dyes are diffusely distributed in cells, others selectively bind to certain cell structures or even to certain chemicals.

To carry out this type of microscopy, special luminescent (fluorescent) microscopes , differing from a conventional light microscope by the presence of a powerful light source (ultra-high pressure mercury-quartz lamp or halogen incandescent quartz lamp), emitting predominantly in the long-wave ultraviolet or short-wave (blue-violet) region of the visible spectrum.

This source is used to excite fluorescence before the light it emits passes through a special exciting (blue-violet) light filter and is reflected interference beam splitter record , almost completely cutting off longer wavelength radiation and transmitting only that part of the spectrum that excites fluorescence. At the same time, in modern models of fluorescent microscopes, exciting radiation hits the specimen through the lens (!) After excitation of fluorescence, the resulting light again enters the lens, after which it passes through the one located in front of the eyepiece locking (yellow) light filter , cutting off short-wave exciting radiation and transmitting luminescence light from the drug to the observer's eye.

Due to the use of such a system of light filters, the luminescence intensity of the observed object is usually low, and therefore fluorescent microscopy should be carried out in special darkened rooms .

An important requirement when performing this type of microscopy is also the use non-fluorescent immersion And enclosing media . In particular, to quench the intrinsic fluorescence of cedar or other immersion oil, small amounts of nitrobenzene are added to it (from 2 to 10 drops per 1 g). In turn, a buffer solution of glycerol, as well as non-fluorescent polymers (polystyrene, polyvinyl alcohol) can be used as containing media for drugs. Otherwise, when performing luminescence microscopy, ordinary glass slides and coverslips are used, which transmit radiation in the used part of the spectrum and do not have their own luminescence.

Accordingly, important advantages of fluorescence microscopy are:

1) color image;

2) high degree of contrast of self-luminous objects on a black background;

3) the possibility of studying cellular structures that selectively absorb various fluorochromes, which are specific cytochemical indicators;

4) the ability to determine functional and morphological changes in cells in the dynamics of their development;

5) the possibility of specific staining of microorganisms (using immunofluorescence).

Electron microscopy

The theoretical foundations for using electrons to observe microscopic objects were laid W. Hamilton , who established an analogy between the passage of light rays in optically inhomogeneous media and the trajectories of particles in force fields, as well as de Broglie , who put forward the hypothesis that the electron has both corpuscular and wave properties.

Moreover, due to the extremely short wavelength of electrons, which decreases in direct proportion to the applied accelerating voltage, the theoretically calculated resolution limit , which characterizes the ability of the device to separately display small, maximally located details of an object, for an electron microscope is 2-3 Å ( Angstrom , where 1Å=10 -10 m), which is several thousand times higher than that of an optical microscope. The first image of an object formed by electron beams was obtained in 1931. German scientists M. Knollem And E. Ruska .

In the designs of modern electron microscopes, the source of electrons is metal (usually tungsten), from which, after heating to 2500 ºС, the result is thermionic emission electrons are emitted. With the help of electric and magnetic fields, the formed electron flow You can speed up and slow down, as well as deflect in any direction and focus. Thus, the role of lenses in an electron microscope is played by a set of appropriately designed magnetic, electrostatic and combined devices called “ electronic lenses" .

A necessary condition for the movement of electrons in the form of a beam over a long distance is also the creation of vacuum , since in this case the average free path of electrons between collisions with gas molecules will significantly exceed the distance over which they must move. For these purposes, it is sufficient to maintain a negative pressure of approximately 10 -4 Pa in the working chamber.

According to the nature of studying objects, electron microscopes are divided into translucent, reflective, emissive, raster, shadow And mirrored , among which the first two are the most commonly used.

Optical design transmission (transmission) electron microscope is entirely equivalent to the corresponding optical microscope design in which the light beam is replaced by an electron beam and glass lens systems are replaced by electron lens systems. Accordingly, a transmission electron microscope consists of the following main components: lighting system, object camera, focusing system And final image registration block , consisting of a camera and a fluorescent screen.

All these nodes are connected to each other, forming a so-called “microscope column”, inside which a vacuum is maintained. Another important requirement for the object under study is its thickness of less than 0.1 microns. The final image of the object is formed after appropriate focusing of the electron beam passing through it onto photographic film or fluorescent screen , coated with a special substance - phosphor (similar to the screen in TV picture tubes) and turning the electronic image into a visible one.

In this case, the formation of an image in a transmission electron microscope is associated mainly with different degrees of electron scattering by different areas of the sample under study and, to a lesser extent, with differences in electron absorption by these areas. Contrast is also enhanced by using “ electronic dyes "(osmium tetroxide, uranyl, etc.), selectively binding to certain areas of the object. Modern transmission electron microscopes, designed in a similar way, provide maximum useful magnification up to 400,000 times, which corresponds to resolution at 5.0 Å. The fine structure of bacterial cells revealed using transmission electron microscopy is called ultrastructure .

IN reflective (scanning) electron microscope the image is created using electrons reflected (scattered) by the surface layer of an object when it is irradiated at a small angle (approximately a few degrees) to the surface. Accordingly, the formation of an image is due to the difference in electron scattering at different points of an object depending on its surface microrelief, and the result of such microscopy itself appears in the form of the structure of the surface of the observed object. Contrast can be enhanced by sputtering metal particles onto the surface of the object. The achieved resolution of microscopes of this type is about 100 Å.

Designed to form enlarged two-dimensional images taken in focal planes of the sample sequentially located along the optical axis, which provides the possibility of two- and three-dimensional examination of small structural details of the sample. The optical components are mounted on a durable, ergonomic base, allowing for quick replacement, precise centering and careful alignment of optically interconnected assemblies. Together, the optical and mechanical components of the microscope, including the sample placed between the slide and coverslip, form an optical system whose central axis passes through the base and stand of the microscope.

The optical system of a microscope usually consists of an illuminator (including a light source and a collecting lens), a condenser, a sample, an objective, an eyepiece, and a photodetector, which can be either a camera or the observer's eye. Research microscopes also contain a light beam (pre-)processing device, usually located between the illuminator and the condenser, and an additional photodetector or filters inserted between the lens and the eyepiece or camera. The coordinated operation of the photodetector and beam pre-processing device(s) provides changes in image contrast as a function of spatial frequency, phase, polarization, absorption, fluorescence, off-axis illumination and/or other sample properties and lighting conditions. But even without additional devices for processing the illumination beam and filtering the waves that form the image, most even basic microscopic configurations have a certain degree of natural filtering.

Introduction

Modern complex microscopes are designed to form enlarged two-dimensional images taken in focal planes of the sample sequentially located along the optical axis, which provides the possibility of two and three-dimensional examination of small structural details of the sample.

Most microscopes are equipped with a stage movement mechanism that allows the microscopist to precisely position, orient, and focus the specimen to optimize observation and imaging. The intensity of illumination and the path of rays in a microscope are controlled and controlled by placing diaphragms, mirrors, prisms, beam splitters and other optical elements in certain positions, thereby achieving the required brightness and contrast of the sample.

Figure 1 shows a Nikon Eclipse E600 microscope, with a trinocular tube and a DXM-1200 digital camera for image recording. Illumination is produced by a halogen lamp with a tungsten filament located in the lamp unit, the light from which first passes through a collecting lens and then enters the optical path at the base of the microscope. The beam of light emitted by the incandescent lamp is modified by a series of filters also located at the base of the microscope, after which, reflected from the mirror, it falls through the field diaphragm onto the condenser. The cone of light formed by the condenser illuminates the sample located on the microscope stage and enters the objective. After the lens, the light beam is split by a beam splitter/prism unit and directed either to the eyepiece, where a virtual image is formed, or to the projection lens of the trinocular intermediate tube to form a digital image on the CCD photodiode matrix of the digital image recording and visualization system.

The optical components of modern microscopes are mounted on a durable ergonomic base, which allows for quick replacement, precise centering and careful adjustment of optically interconnected assemblies. Together, the optical and mechanical components of the microscope, including the sample placed between the slide and coverslip, form an optical system whose central axis passes through the base and stand of the microscope.

Microscope optical system usually consists of an illuminator (including a light source and a collecting lens), a condenser, a sample, a lens, an eyepiece, and a photodetector, which can be either a camera or the observer's eye (Table 1).
Research microscopes also contain a light beam pre-processing device, usually located between the illuminator and the condenser, and an additional photodetector or light filters placed between the lens and the eyepiece or camera. The coordinated operation of the photodetector and beam pre-processing device(s) provides changes in image contrast as a function of spatial frequency, phase, polarization, absorption, fluorescence, off-axis illumination and/or other sample properties and lighting conditions. But even without additional devices for processing the illumination beam and filtering the waves that form the image, most basic microscopic configurations have a certain degree of natural filtering.

Table 1. Components of the microscope optical system.

Microscope component	Elements and characteristics
Illuminator	Light source, converging lens, field diaphragm, thermal filters, leveling filters, diffuser, neutral density filters
Beam pre-processing device	Condenser iris diaphragm, dark field diaphragm, shadow mask, phase rings, off-axis slit diaphragm, Nomarski prism, fluorescence excitation filter
Condenser	Numerical aperture, focal length, aberrations, light transmission, immersion medium, working distance
Sample	Slide thickness, coverslip thickness, immersion medium, absorption, transmission, diffraction, fluorescence, retardation, birefringence
Lens	Magnification, numerical aperture, focal length, immersion medium, aberrations, light transmission, optical transfer function, working distance
Image filter	Compensator, analyzer, Nomarski prism, lens iris, phase plate, SSEE filter, modulation plate, light transmission, wavelength selection, fluorescence cut-off filter
Eyepiece	Magnification, aberrations, field size, eye offset
Detector	Human eye, photoemulsion, photomultiplier, photodiode matrix, video camera

While some optical components of the microscope act as image-forming elements, others are designed for various modifications of the illuminating beam, and also perform filtering and transmitting functions. The image-forming components of a microscope's optical system are the converging lens (located in or adjacent to the illuminator), the condenser, the objective, the eyepiece tube (or eyepiece), and the refractive elements of the human eye or camera lens. Although some of these components are not typically image-forming components, their characteristics are of paramount importance in determining the quality of the final microscopic image.

Path of light waves through an ideal lens

Understanding the role of the individual lenses that make up the components of an optical system is fundamental to understanding the imaging process in a microscope. The simplest image-forming element is an ideal lens (Figure 2) - ideally corrected, free from aberrations and collecting light into one point. A parallel, paraxial beam of light, refracted in a collecting lens, is focused at its focal point or focus (in Figure 2 it is indicated by the inscription Focus). Such lenses are often called positive, since they promote faster convergence of the convergent (converging) light beam and slow down the divergence of the diverging beam. Light from a point source located at the focal point of the lens emerges from it in a parallel, paraxial beam (direction from right to left in Figure 2). The distance between the lens and its focus is called focal length lenses (indicated by f in Figure 2).

Optical phenomena are often described in terms of either quantum theory or wave optics, depending on the problem at hand. When light passes through a lens, its wave properties can be neglected and it can be assumed to travel in straight lines, usually called rays. Simple ray diagrams or ray paths are often sufficient to explain many important aspects and concepts of microscopy, including refraction, focal length, magnification, imaging, and apertures. In other cases, light waves are more conveniently thought of as consisting of individual particles (quanta), especially when light is created by a quantum mechanical event or transformed into another form of energy. In our discussion, paraxial rays passing through optical lenses will be considered in terms of both wave and geometric (ray) optics (ray diagrams in which rays propagate from left to right). Paraxial (or paraxial) are light rays passing close to the optical axis; in this case, the values ​​of the angles of incidence and refraction, expressed in radians, can be considered approximately equal to the values ​​of their sines.

In a parallel light beam, individual monochromatic waves form group of waves, electric and magnetic vectors in which oscillate in phase and form wave front; in this case, the direction of its propagation is perpendicular to the direction of oscillations. When passing through an ideal lens, a plane wave is transformed into a spherical one, centered at the focal point ( Focus) lenses (Figure 2). Light waves brought together at a focal point interfere, reinforcing each other. Conversely, a spherical wave front diverging from the focal point of an ideal lens is transformed by it into a plane wave (propagation from right to left in Figure 2). Each light ray of a plane wave is refracted in a lens with a slight difference from the others because it hits its surface at a slightly different angle. At the exit from the lens, the direction of the light beam also changes. In real systems, the refractive angle and focal point of a lens or group of lenses depend on the thickness, geometry, refractive index, and dispersion of each component of the system.

Electrical part of the microscope

Unlike a magnifying glass, a microscope has at least two magnification levels. The functional and structural and technological parts of the microscope are designed to ensure the operation of the microscope and obtain a stable, most accurate, enlarged image of the object. Here we will look at the structure of a microscope and try to describe the main parts of the microscope.

Functionally, the microscope device is divided into 3 parts:

1. Lighting part

The lighting part of the microscope design includes a light source (lamp and electrical power supply) and an optical-mechanical system (collector, condenser, field and aperture adjustable/iris diaphragms).

2. Reproducing part

Designed to reproduce an object in the image plane with the image quality and magnification required for research (i.e., to construct an image that would reproduce the object as accurately as possible and in all details with the resolution, magnification, contrast and color rendition corresponding to the microscope optics).
The reproducing part provides the first stage of magnification and is located after the object to the microscope image plane.
The reproducing part includes a lens and an intermediate optical system.

Modern microscopes of the latest generation are based on optical lens systems corrected for infinity. This additionally requires the use of so-called tube systems, which “collect” parallel beams of light emerging from the lens in the microscope image plane.

3. Visualization part

Designed to obtain a real image of an object on the retina of the eye, photographic film or plate, on the screen of a television or computer monitor with additional magnification (second stage of magnification).
The imaging part is located between the image plane of the lens and the eyes of the observer (digital camera).
The imaging part includes a monocular, binocular or trinocular visual attachment with an observation system (eyepieces that work like a magnifying glass).
In addition, this part includes additional magnification systems (magnification wholesaler/change systems); projection attachments, including discussion attachments for two or more observers; drawing apparatus; image analysis and documentation systems with appropriate adapters for digital cameras.

Layout of the main elements of an optical microscope

From a design and technological point of view, the microscope consists of the following parts:

• mechanical;
• optical;
• electric.

1. Mechanical part of the microscope

Microscope device turns on itself tripod, which is the main structural and mechanical block of the microscope. The tripod includes the following main blocks: base And tube holder.

Base is a block on which the entire microscope is mounted and is one of the main parts of the microscope. In simple microscopes, lighting mirrors or overhead illuminators are installed on the base. In more complex models, the lighting system is built into the base without or with a power supply.

Types of microscope bases:

1. base with lighting mirror;
2. so-called “critical” or simplified lighting;
3. Koehler lighting.

1. a lens changing unit, which has the following design options - a revolving device, a threaded device for screwing in a lens, a “sled” for threadless mounting of lenses using special guides;
2. focusing mechanism for coarse and fine adjustment of the microscope for sharpness - mechanism for focusing movement of lenses or stages;
3. attachment point for replaceable object tables;
4. mounting unit for focusing and centering movement of the condenser;
5. attachment point for replaceable attachments (visual, photographic, television, various transmitting devices).

Microscopes may use stands to mount components (for example, a focusing mechanism in stereo microscopes or an illuminator mount in some models of inverted microscopes).

The purely mechanical component of the microscope is stage, intended for fastening or fixing an observation object in a certain position. Tables can be fixed, coordinated and rotating (centered and non-centered).

2. Microscope optics (optical part)

Optical components and accessories provide the main function of the microscope - creating an enlarged image of an object with a sufficient degree of reliability in shape, size ratio of the constituent elements and color. In addition, the optics must provide an image quality that meets the objectives of the study and the requirements of the analysis methods.
The main optical elements of a microscope are the optical elements that form the lighting (including the condenser), observation (eyepieces) and reproducing (including lenses) systems of the microscope.

Microscope Objectives

— are optical systems designed to construct a microscopic image in the image plane with appropriate magnification, element resolution, and accuracy of reproduction of the shape and color of the object of study. Objectives are one of the main parts of a microscope. They have a complex optical-mechanical design, which includes several single lenses and components glued together from 2 or 3 lenses.
The number of lenses is determined by the range of tasks solved by the lens. The higher the image quality that a lens produces, the more complex its optical design. The total number of lenses in a complex objective can be up to 14 (for example, this could apply to a planochromatic objective with a magnification of 100x and a numerical aperture of 1.40).

The lens consists of front and rear parts. The front lens (or lens system) faces the specimen and is the main one in constructing an image of appropriate quality; it determines the working distance and numerical aperture of the lens. The subsequent part, in combination with the front part, provides the required magnification, focal length and image quality, and also determines the height of the lens and the length of the microscope tube.

Lens classification

The classification of lenses is much more complicated than the classification of microscopes. Lenses are divided according to the principle of calculated image quality, parametric and design-technological characteristics, as well as according to research and contrast methods.

According to the principle of calculated image quality lenses can be:

• achromatic;
• apochromatic;
• flat field lenses (plan).

Achromatic lenses.

Achromatic lenses are designed for use in the spectral range 486-656 nm. Correction of any aberration (achromatization) is performed for two wavelengths. These lenses eliminate spherical aberration, chromatic position aberration, coma, astigmatism and partially spherochromatic aberration. The image of the object has a slightly bluish-reddish tint.

Apochromatic lenses.

Apochromatic objectives have an extended spectral region and achromatization is performed at three wavelengths. At the same time, in addition to position chromatism, spherical aberration, coma and astigmatism, the secondary spectrum and spherochromatic aberration are also corrected quite well, thanks to the introduction of crystal lenses and special glasses into the design. Compared to achromat lenses, these lenses typically have higher numerical apertures, produce sharper images, and accurately reproduce the color of the subject.

Semi-apochromats or microfluars.

Modern lenses with intermediate image quality.

Planlenses.

In plan lenses, the curvature of the image across the field has been corrected, which ensures a sharp image of the object throughout the entire observation field. Plan lenses are usually used in photography, with plan apochromats being the most effective.

The need for this type of lenses is increasing, but they are quite expensive due to the optical design that implements a flat image field and the optical media used. Therefore, routine and work microscopes are equipped with so-called economical lenses. These include lenses with improved image quality across the field: achromats (LEICA), CP achromats and achroplanes (CARL ZEISS), stigmachromats (LOMO).

According to parametric characteristics lenses are divided as follows:

1. objectives with a finite tube length (for example, 160 mm) and objectives corrected for the tube length “infinity” (for example, with an additional tube system having a microscope focal length of 160 mm);
2. small lenses (up to 10x); medium (up to 50x) and high (more than 50x) magnifications, as well as lenses with ultra-high magnification (over 100x);
3. lenses of small (up to 0.25), medium (up to 0.65) and large (more than 0.65) numerical apertures, as well as lenses with increased (compared to conventional) numerical apertures (for example, apochromatic correction lenses, as well as special lenses for fluorescent microscopes);
4. lenses with increased (compared to conventional) working distances, as well as with large and extra-long working distances (lenses for working in inverted microscopes). The working distance is the free distance between the object (the plane of the cover glass) and the lower edge of the frame (lens, if it protrudes) of the front component of the lens;
5. lenses that provide observation within the normal linear field (up to 18 mm); wide-field lenses (up to 22.5 mm); ultra-wide-field lenses (over 22.5 mm);
6. lenses are standard (45 mm, 33 mm) and non-standard in height.

Height - the distance from the reference plane of the lens (the plane of contact of the screwed-in lens with the revolving device) to the plane of the object with a focused microscope, is a constant value and ensures the parfocality of a set of similar height lenses of different magnifications installed in the revolving device. In other words, if you use a lens of one magnification to obtain a sharp image of an object, then when moving to subsequent magnifications, the image of the object remains sharp within the depth of field of the lens.

According to design and technological characteristics there is the following division:

1. lenses with a spring frame (starting from numerical aperture 0.50) and without it;
2. lenses that have an iris diaphragm inside to change the numerical aperture (for example, in lenses with an increased numerical aperture, in transmitted light lenses to implement the dark field method, in reflected light polarized lenses);
3. lenses with a corrective (control) frame, which ensures the movement of optical elements inside the lens (for example, to adjust the image quality of the lens when working with different cover glass thicknesses or with different immersion liquids; as well as to change the magnification during a smooth - pancratic - change in magnification) and without her.

To provide research and contrasting methods lenses can be divided as follows:

1. objectives working with and without cover glass;
2. lenses of transmitted and reflected light (non-reflex); luminescent lenses (with a minimum of intrinsic luminescence); polarized lenses (without glass tension in the optical elements, i.e., without introducing their own depolarization); phase lenses (having a phase element - a translucent ring inside the lens); DIC lenses operating using the differential interference contrast method (polarizing with a prism element); epilenses (reflected light lenses, designed to provide light and dark field methods, have specially designed lighting epi-mirrors in their design);
3. immersion and non-immersion lenses.

Immersion ( from lat. immersio - immersion) is a liquid that fills the space between the object of observation and a special immersion objective (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 a microscope or its use is required by the technological process of microscopy. This happens:

1. increasing visibility by increasing the difference between 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.

Immersion lenses. The image quality, parameters and optical design of immersion lenses are calculated and selected taking into account the thickness of the immersion layer, which is considered as an additional lens with a corresponding refractive index. Immersion liquid placed between the object and the front component of the lens increases the angle at which the object is viewed (aperture angle). The numerical aperture of an immersion-free (dry) lens does not exceed 1.0 (resolution is about 0.3 µm for the main wavelength); immersion - reaches 1.40 depending on the refractive index of immersion and the technological capabilities of manufacturing the front lens (the resolution of such a lens is about 0.12 microns).
High magnification immersion objectives have a short focal length of 1.5-2.5 mm with a free working distance of 0.1-0.3 mm (the distance from the plane of the specimen to the frame of the front lens of the lens).

Lens markings.

Data about each lens is marked on its body indicating the following parameters:

1. magnification (“x”-fold, times): 8x, 40x, 90x;
2. NA: 0.20; 0.65, example: 40/0.65 or 40x/0.65;
3. additional letter marking if the lens is used for various research and contrast methods: phase - Ф (Рп2 - the number corresponds to the marking on a special condenser or insert), polarizing - П (Pol), luminescent - Л (L), phase-luminescent - FL ( PhL), EPI (Epi, HD) - epilens for working in reflected light using the dark field method, differential interference contrast - DIC (DIC), example: 40x/0.65 F or Ph2 40x/0.65;
4. marking of the type of optical correction: apochromat - APO (APO), planchromat - PLAN (PL, Plan), planachromat - PLAN-APO (Plan-Aro), improved achromat, semi-plan - CX - stigmachromat (Achrostigmat, CP-achromat, Achroplan), microfluar (semiplan-semi-apochromat) - SF or M-FLUAR (MICROFLUAR, NEOFLUAR, NPL, FLUOTAR).

Eyepieces

Optical systems designed to construct a microscopic image on the retina of the observer's eye. In general, eyepieces consist of two groups of lenses: the eye lens - closest to the observer’s eye - and the field lens - closest to the plane in which the lens builds an image of the object in question.

Eyepieces are classified according to the same groups of characteristics as lenses:

1. eyepieces with compensatory (K - compensate for the chromatic difference in lens magnification over 0.8%) and non-compensatory action;
2. regular and flat field eyepieces;
3. wide-angle eyepieces (with an eyepiece number - the product of the magnification of the eyepiece and its linear field - more than 180); ultra-wide-angle (with an ocular number of more than 225);
4. eyepieces with extended pupil for working with or without glasses;
5. observation eyepieces, projection eyepieces, photo eyepieces, gamals;
6. eyepieces with internal aiming (using a moving element inside the eyepiece, adjustment is made to a sharp image of the reticle or the microscope image plane; as well as a smooth, pancratic change in the magnification of the eyepiece) and without it.

Lighting system

The lighting system is an important part microscope designs and is a system of lenses, diaphragms and mirrors (the latter are used if necessary), ensuring uniform illumination of the object and complete filling of the lens aperture.
The illumination system of a transmitted light microscope consists of two parts: a collector and a condenser.

Collector.
With a built-in transmitted light illumination system, the collector part is located near the light source at the base of the microscope and is designed to increase the size of the luminous body. To ensure adjustment, the collector can be made movable and move along the optical axis. The field diaphragm of the microscope is located near the collector.

Condenser.
The condenser's optical system is designed to increase the amount of light entering the microscope. The condenser is located between the object (stage) and the illuminator (light source).
Most often, in educational and simple microscopes, the condenser can be made non-removable and motionless. In other cases, the condenser is a removable part and, when adjusting the lighting, has a focusing movement along the optical axis and a centering movement perpendicular to the optical axis.
At the condenser there is always a lighting aperture iris diaphragm.

The condenser is one of the main elements that ensures the operation of the microscope using various methods of illumination and contrast:

• oblique illumination (diaphragm from the edge to the center and displacement of the lighting aperture diaphragm relative to the optical axis of the microscope);
• dark field (maximum aperture from the center to the edge of the lighting aperture);
• phase contrast (ring illumination of an object, while the image of the light ring fits into the phase ring of the lens).

Classification of capacitors is close in groups of characteristics to lenses:

1. Condensers based on image quality and type of optical correction are divided into non-achromatic, achromatic, aplanatic and achromatic-aplanatic;
2. condensers of small numerical aperture (up to 0.30), medium numerical aperture (up to 0.75), large numerical aperture (over 0.75);
3. condensers with regular, long and extra-long working distances;
4. conventional and special condensers for various research and contrast methods;
5. The condenser design is single, with a folding element (front component or large-field lens), with a screw-on front element.

Abbe condenser- a condenser not corrected for image quality, consisting of 2 non-achromatic lenses: one is biconvex, the other is plano-convex, facing the object of observation (the flat side of this lens is directed upward). Condenser aperture, A = 1.20. Has an iris diaphragm.

Aplanatic condenser- a condenser consisting of three lenses arranged as follows: the top lens is plano-convex (the flat side is directed towards the lens), followed by concave-convex and biconvex lenses. Corrected regarding spherical aberration and coma. Condenser aperture, A = 1.40. Has an iris diaphragm.

Achromatic condenser- condenser fully corrected for chromatic and spherical aberration.

Dark field condenser- a condenser designed to obtain a dark field effect. It can be special or converted from a regular bright-field condenser by installing an opaque disk of a certain size in the plane of the iris diaphragm of the condenser.

Condenser marking.
The numerical aperture (illumination) is marked on the front of the condenser.

3. Electrical part of the microscope

Modern microscopes, instead of mirrors, use various lighting sources powered from an electrical network. These can be either ordinary incandescent lamps, or halogen, xenon, or mercury lamps. LED lighting is also becoming increasingly popular. They have significant advantages over conventional lamps, such as durability, lower energy consumption, etc. To power the lighting source, various power supplies, ignition units and other devices are used that convert the current from the electrical network into one suitable for powering a particular lighting source. These can also be rechargeable batteries, which allows you to use microscopes in the field in the absence of a connection point.