Resolution limit- this is the smallest distance between two points of an object at which these points are distinguishable, i.e. are perceived in a microscope as two points.

Resolution is defined as the ability of a microscope to produce separate images of small details of the object being examined. It is given by the formula:

where A is the numerical aperture, l is the wavelength of light; , where n is the refractive index of the medium in which the object in question is located, U is the aperture angle.

To study the structure of the smallest living creatures, microscopes with high magnification and good resolution are needed. An optical microscope is limited to a magnification of 2000 times and has a resolution of no better than 250 nm. These values ​​are not suitable for studying fine details of cells.

118. Ultraviolet microscope. One way to reduce

The limit of microscope resolution is the use of light with a shorter wavelength. In this regard, an ultraviolet microscope is used, in which microobjects are examined in ultraviolet rays. Since the eye does not directly perceive this radiation, photographic plates, fluorescent screens or electro-optical converters are used. Another way to reduce the resolution limit of a microscope is to increase the refractive index of the medium in which the microscope is located. To do this, it is placed in immersion liquid, for example, cedar oil.

119. Luminescent (fluorescent) microscopy is based on the ability of some substances to luminesce, that is, to glow when illuminated with invisible ultraviolet or blue light.

The luminescence color is shifted to a longer wavelength part of the spectrum compared to the light that excites it (Stokes' rule). When luminescence is excited by blue light, its color can range from green to red; if luminescence is excited by ultraviolet radiation, then the luminescence can be in any part of the visible spectrum. This feature of luminescence allows, using special filters that absorb exciting light, to observe a relatively weak luminescent glow.

Since most microorganisms do not have their own luminescence, they are stained with solutions of fluorescent dyes. This method is used for bacterioscopic examination of the causative agents of certain infections: tuberculosis (auromine), inclusions in cells formed by certain viruses, etc. The same method can be used for the cytochemical study of living and fixed microorganisms. In the immunofluorescence reaction using antibodies labeled with fluorochromes, antigens of microorganisms or antibodies are detected in the serum of patients

120. Phase contrast microscopy. When microscopying unstained microorganisms other than environment only according to the refractive index, there is no change in light intensity (amplitude), but only the phase of the transmitted light waves changes. Therefore, the eye cannot notice these changes and the observed objects look low-contrast and transparent. To observe such objects use phase contrast microscopy, based on the transformation of invisible phase changes introduced by an object into amplitude changes visible to the eye.

Thanks to the use of this method of microscopy, the contrast of living unstained microorganisms increases dramatically and they appear dark on a light background or light on a dark background.

Phase contrast microscopy is also used to study tissue culture cells, observe the effects of various viruses on cells, etc.

121. Dark-field microscopy. Dark-field microscopy is based on the ability of microorganisms to strongly scatter light. For dark-field microscopy, conventional objectives and special dark-field condensers are used.

The main feature of dark-field condensers is that their central part is darkened and direct rays from the illuminator do not enter the microscope lens. The object is illuminated by oblique side rays and only rays scattered by particles in the preparation enter the microscope lens. Dark-field microscopy is based on the Tyndall effect, a famous example of which is the detection of dust particles in the air when illuminated by a narrow beam of sunlight.

With dark-field microscopy, microorganisms appear brightly glowing against a black background. With this method of microscopy, the smallest microorganisms can be detected, the sizes of which are beyond the resolution of the microscope. However, dark-field microscopy allows you to see only the outlines of an object, but does not allow you to study the internal structure.

122. Thermal radiation is the most common type of electromagnetic radiation in nature. It occurs due to the energy of thermal motion of atoms and molecules of a substance. Thermal radiation is inherent in all bodies at any temperature other than absolute zero.

Total body emissivity E (also called energetic luminosity) is the amount of energy emitted from a unit surface area of ​​a body in 1 s. Measured in J/m 2 s.

Total radiation absorption capacity of the body A (absorption coefficient) is the ratio of radiant energy absorbed by a body to all radiant energy incident on it; A is a dimensionless quantity.

123. Absolutely black body. An imaginary body that absorbs all radiant energy incident on it at any temperature is called absolutely black.

Kirchhoff's law. For all bodies at a given temperature, the ratio of emissivity E to radiation absorption ability A is a constant value equal to the emissivity of an absolutely black body e at the same temperature:

e.

Stefan-Boltzmann law. The total emissivity of a black body is directly proportional to the fourth power of its absolute temperature:

e=sT 4 ,

where s is the Stefan-Boltzmann constant.

Wine's Law. The wavelength corresponding to the maximum radiation of a black body is inversely proportional to its absolute temperature:

l t ×T = V,

where v is Wien’s constant.

Based on the Law of Wine optical pyrometry– a method for determining the temperature of hot bodies (metal in a smelting furnace, gas in a cloud of an atomic explosion, the surface of stars, etc.) from their radiation spectrum. It was this method that first determined the temperature of the surface of the Sun.

124 . Infrared radiation. Electromagnetic radiation that occupies the spectral region between the red limit of visible light (λ = 0.76 μm) and short-wave radio radiation (λ = 1 - 2 mm) is called infrared (IR). Heated solids and liquids emit a continuous infrared spectrum.

The therapeutic use of infrared radiation is based on its thermal effect. Special lamps are used for treatment.

Infrared radiation penetrates the body to a depth of about 20 mm, so the surface layers are heated to a greater extent. The therapeutic effect is due to the resulting temperature gradient, which activates the activity of the thermoregulatory system. Increasing the blood supply to the irradiated area leads to favorable therapeutic consequences.

125. Ultraviolet radiation. Electromagnetic radiation,

occupying the spectral region between the violet edge of visible light (λ = 400 nm) and the long-wave part of X-ray radiation (λ = 10 nm) is called ultraviolet (UV).

Heated solids at high temperatures emit

a significant amount of ultraviolet radiation. However, the maximum

The spectral density of energetic luminosity, in accordance with Wien's law, is at 7000 K. In practice, this means that under normal conditions the thermal radiation of gray bodies cannot serve as an effective source of UV radiation. The most powerful source of UV radiation is the Sun, 9% of whose radiation at the boundary of the earth's atmosphere is ultraviolet.

UV radiation is necessary for the operation of UV microscopes, fluorescent microscopes, and for fluorescent analysis. The main use of UV radiation in medicine is associated with its specific biological effects, which are caused by photochemical processes.

126. Thermography– this is the registration of radiation from various areas

body surface for the purpose of diagnostic interpretation. Temperature is determined in two ways. In one case, liquid crystal displays are used, the optical properties of which are very sensitive to small changes in temperature.

By placing these indicators on the patient's body, it is possible to visually determine the local temperature difference by changing their color.

Another method is based on using thermal imagers, which use sensitive infrared radiation detectors, such as photoresistors.

127. Physiological basis of thermography. Physiological processes occurring in the human body are accompanied by the release of heat, which is transferred by circulating blood and lymph. The source of heat is biochemical processes occurring in a living organism. The heat generated is carried by the blood throughout the body. Possessing high heat capacity and thermal conductivity, circulating blood is capable of intense heat exchange between the central and peripheral regions of the body. The temperature of the blood passing through the skin vessels decreases by 2-3°.

Thermography is based on the phenomenon of an increase in the intensity of infrared radiation over pathological foci (due to increased blood supply and metabolic processes in them) or a decrease in its intensity in areas with reduced regional blood flow and accompanying changes in tissues and organs. This is usually expressed by the appearance of a "hot zone". There are two main types of thermography: telethermography and contact cholesteric thermography.

128. Telethermography is based on the conversion of infrared radiation from the human body into an electrical signal, which is visualized on the screen of a thermal imager. Sensitive photoresistors are used as receiving devices for infrared radiation in thermal imagers.

The thermal imager works as follows. Infrared radiation is focused by a lens system and then hits a photodetector, which operates when cooled to –196°C. The signal from the photodetector is amplified and subjected to digital processing with subsequent transmission of the received information to the screen of a color monitor.

129. Contact liquid crystal thermography relies on the optical properties of anisotropic cholesteric liquid crystals, which manifest themselves as a change in color to rainbow colors when applied to thermally emitting surfaces. The coldest areas are red, the hottest are blue.

Liquid crystal contact plate thermography is currently widely and successfully used in various fields of medicine, but remote methods for recording infrared radiation of the human body have found much greater use.

130. Clinical applications of thermography. Thermographic diagnostics do not cause any external impact or inconvenience to the patient and allow you to “see” the abnormalities in the thermal pattern on the surface of the patient’s skin, which are characteristic of many diseases and physical disorders.

Thermography, being a physiological, harmless, non-invasive diagnostic method, finds its use in practical medicine for diagnosing a wide range of pathologies: diseases of the mammary glands, spine, joints, thyroid gland, ENT organs, blood vessels, liver, gall bladder, intestines, stomach, pancreas , kidneys, bladder, prostate gland. Thermography allows you to record changes at the very beginning of the development of the pathological process, before the appearance of structural changes in tissues.

131. Rutherford (planetary) model of the atom. According to this model, all the positive charge and almost all the mass (more than 99.94%) of an atom are concentrated in the atomic nucleus, the size of which is negligible (about 10 -13 cm) compared to the size of the atom (10 -8 cm). Electrons move around the nucleus in closed (elliptical) orbits, forming the electron shell of the atom. The charge of the nucleus is equal in absolute value to the total charge of the electrons.

Disadvantages of the Rutherford model.

a) in the Rutherford model the atom is unstable

education, while experience indicates the opposite;

b) according to Rutherford, the radiation spectrum of an atom is continuous, while experience speaks of the discrete nature of the radiation.

132. Quantum theory of the structure of the atom according to Bohr. Based on the idea of ​​the discreteness of the energy states of the atom, Bohr improved Rutherford's atomic model, creating a quantum theory of the structure of the atom. It is based on three postulates.

Electrons in an atom cannot move in any orbits, but only in orbits of a very certain radius. In these orbits, called stationary, the angular momentum of the electron is determined by the expression:

where m is the mass of the electron, v is its speed, r is the radius of the electron orbit, n is an integer called quantum (n=1,2,3, ...).

The movement of electrons in stationary orbits is not accompanied by radiation (absorption) of energy.

Transfer of an electron from one stationary orbit to another

accompanied by the emission (or absorption) of an energy quantum.

The value hn of this quantum is equal to the energy difference W 1 – W 2 of the stationary states of the atom before and after radiation (absorption):

hn=W 1 – W 2.

This relationship is called the frequency condition.

133. Types of spectra. There are three main types of spectra: solid, line and striped.

Line spectra

atoms. Emission is caused by transitions of bound electrons to lower energy levels.

Striped spectra are emitted by individual excited

molecules. Radiation is caused by electronic transitions in atoms, and by the vibrational movements of the atoms themselves in the molecule.

Continuous spectra emitted by collections of many molecular and atomic ions interacting with each other.

The main role in radiation is played by the chaotic movement of these particles, caused by high temperature.

134. Concept of spectral analysis. Every chemical element

emits (and absorbs) light with very specific wavelengths unique to this element. Line spectra of elements are obtained by photographing in spectrographs in which light is decomposed using a diffraction grating. The line spectrum of an element is a kind of “fingerprint” that allows you to accurately identify this element based on the wavelengths of emitted (or absorbed) light. Spectrographic studies are one of the most powerful chemical analysis techniques available to us.

Qualitative spectral analysis– this is a comparison of the obtained spectra with the tabulated ones to determine the composition of the substance.

Quantitative spectral analysis carried out by photometry (determining the intensity) of spectral lines: the brightness of the lines is proportional to the amount of a given element.

Spectroscope calibration. In order to use a spectroscope to determine the wavelengths of the spectrum under study, the spectroscope must be calibrated, i.e. establish the relationship between the wavelengths of spectral lines and the divisions of the spectroscope scale at which they are visible.

135. Main characteristics and areas of application of spectral analysis. Using spectral analysis, you can determine both the atomic and molecular composition of a substance. Spectral analysis allows for the qualitative discovery of individual components of the analyzed sample and the quantitative determination of their concentration. Substances with very close chemical properties, which are difficult or even impossible to analyze by chemical methods, are easily determined spectrally.

Sensitivity spectral analysis is usually very high. Direct analysis achieves a sensitivity of 10 -3 - 10 -6%. Speed Spectral analysis usually significantly exceeds the speed of analysis performed by other methods.

136. Spectral analysis in biology. The spectroscopic method of measuring the optical activity of substances is widely used to determine the structure of biological objects. When studying biological molecules, their absorption spectra and fluorescence are measured. Dyes that fluoresce under laser excitation are used to determine the hydrogen index and ionic strength in cells, as well as to study specific areas in proteins. Using resonant Raman scattering, the structure of cells is probed and the conformation of protein and DNA molecules is determined. Spectroscopy played an important role in the study of photosynthesis and the biochemistry of vision.

137. Spectral analysis in medicine. There are more than eighty chemical elements in the human body. Their interaction and mutual influence ensures the processes of growth, development, digestion, respiration, immunity, hematopoiesis, memory, fertilization, etc.

For the diagnosis of micro- and macroelements, as well as their quantitative imbalance, hair and nails are the most fertile material. Each hair stores integral information about the mineral metabolism of the entire organism over the entire period of its growth. Spectral analysis provides complete information about the mineral balance over a long period of time. Some toxic substances can only be detected using this method. For comparison: conventional methods allow you to determine the ratio of less than ten microelements at the time of testing using a blood test.

The results of spectral analysis help the doctor in diagnosing and searching for the cause of diseases, identifying hidden diseases and predisposition to them; allow you to more accurately prescribe medications and develop individual schemes for restoring mineral balance.

It is difficult to overestimate the importance of spectroscopic methods in pharmacology and toxicology. In particular, they make it possible to analyze samples of pharmacological drugs during their validation, as well as to identify falsified ones. medicines. In toxicology, ultraviolet and infrared spectroscopy allowed the identification of many alkaloids from Stas extracts.

138. Luminescence Excessive radiation of a body at a given temperature, having a duration significantly exceeding the period of the emitted light waves, is called.

Photoluminescence. Luminescence caused by photons is called photoluminescence.

Chemiluminescence. Luminescence accompanying chemical reactions is called chemiluminescence.

139. Luminescent analysis based on observing the luminescence of objects for the purpose of studying them; used to detect the initial stages of food spoilage, sort pharmacological drugs and diagnose certain diseases.

140. Photoelectric effect called the pullout phenomenon

electrons from a substance under the influence of light incident on it.

At external photoelectric effect an electron leaves the surface of a substance.

At internal photoelectric effect the electron is freed from its bonds with the atom, but remains inside the substance.

Einstein's equation:

where hn is the energy of the photon, n is its frequency, A is the work function of the electron, is the kinetic energy of the emitted electron, v is its speed.

Laws of the photoelectric effect:

The number of photoelectrons emitted from the metal surface per unit time is proportional to the light flux incident on the metal.

Maximum initial kinetic energy of photoelectrons

determined by the frequency of the incident light and does not depend on its intensity.

For each metal there is a red limit of the photoelectric effect, i.e. the maximum wavelength l 0 at which the photoelectric effect is still possible.

The external photoelectric effect is used in photomultiplier tubes (PMTs) and electron-optical converters (EOCs). PMTs are used to measure low-intensity light fluxes. With their help, weak bioluminescence can be detected. Image intensifier tubes are used in medicine to enhance the brightness of X-ray images; in thermography – to convert the body’s infrared radiation into visible radiation. In addition, photocells are used in the subway when passing turnstiles, in modern hotels, airports, etc. for automatically opening and closing doors, for automatically turning on and off street lighting, for determining illumination (lux meter), etc.

141. X-ray radiation-This electromagnetic radiation with a wavelength from 0.01 to 0.000001 microns. It causes the phosphor-coated screen to glow and the emulsion to blacken, making it suitable for photography.

X-rays are produced when electrons suddenly stop as they strike the anode in an X-ray tube. First, the electrons emitted by the cathode are accelerated by an accelerating potential difference to speeds of the order of 100,000 km/s. This radiation, called bremsstrahlung, has a continuous spectrum.

The intensity of X-ray radiation is determined by the empirical formula:

where I is the current strength in the tube, U is the voltage, Z is the serial number of the atom of the anticathode substance, k is const.

X-ray radiation resulting from the deceleration of electrons is called “bremsstrahlung”.

Short-wave X-rays are generally more penetrating than long-wave X-rays and are called tough, and long-wave – soft.

At high voltages in the X-ray tube, along with

x-rays having a continuous spectrum produce x-rays having a line spectrum; the latter is superimposed on the continuous spectrum. This radiation is called characteristic, since each substance has its own, characteristic line X-ray spectrum (a continuous spectrum from the anode substance and is determined only by the voltage on the X-ray tube).

142. Properties of X-ray radiation. X-rays have all the properties that characterize light rays:

1) do not deviate in electric and magnetic fields and, therefore, do not carry an electric charge;

2) have a photographic effect;

3) cause gas ionization;

4) capable of causing luminescence;

5) can be refracted, reflected, have polarization and give the phenomenon of interference and diffraction.

143. Moseley's Law. Since atoms of different substances have different energy levels depending on their structure, the spectra of characteristic radiation depend on the structure of the atoms of the anode substance. The characteristic spectra shift toward higher frequencies with increasing nuclear charge. This pattern is known as Moseley's law:

where n is the frequency of the spectral line, Z is the serial number of the emitting element, A and B are constants.

144. Interaction of X-rays with matter. Depending on the ratio of photon energy e and ionization energy A, three main processes take place.

Coherent (classical) scattering. Scattering of long-wave X-rays occurs mainly without changing the wavelength, and is called coherent . It occurs if the photon energy is less than the ionization energy: hn<А. Так как в этом случае энергия фотона рентгеновского излучения и атома не изменяются, то когерентное рассеяние само по себе не вызывает биологического действия.

Incoherent scattering (Compton effect). In 1922 A.Kh. Compton, observing the scattering of hard X-rays, discovered a decrease in the penetrating power of the scattered beam compared to the incident one. This meant that the wavelength of the scattered X-rays was longer than the incident X-rays. Scattering of X-rays with a change in wavelength is called incoherent, and the phenomenon itself is called the Compton effect.

Photo effect. In the photoelectric effect, X-rays are absorbed by an atom, causing an electron to be ejected and the atom to be ionized (photoionization). If the photon energy is insufficient for ionization, then the photoelectric effect can manifest itself in the excitation of atoms without the emission of electrons.

Ionizing effect X-ray radiation manifests itself in an increase in electrical conductivity under the influence of X-rays. This property is used in dosimetry to quantify the effect of this type of radiation.

145. X-ray luminescence called the glow of a number of substances under X-ray irradiation. This glow of platinum-synoxide barium allowed Roentgen to discover the rays. This phenomenon is used to create special luminous screens for the purpose of visual observation of X-rays, sometimes to enhance the effect of X-rays on a photographic plate, which allows these rays to be recorded.

146. X-ray absorption described by Bouguer's law:

F = F 0 e - m x,

where m is the linear attenuation coefficient,

x is the thickness of the substance layer,

F 0 – intensity of incident radiation,

F is the intensity of transmitted radiation.

147. Impact of X-ray radiation on the body. Although radiation exposure during X-ray examinations is small, they can lead to changes in the chromosomal apparatus of cells - radiation mutations. Therefore, X-ray examinations must be regulated.

148. X-ray diagnostics. X-ray diagnostics is based on the selective absorption of X-ray radiation by tissues and organs.

149. X-ray. During fluoroscopy, an image of the transilluminated object is obtained on a fluoroscopic screen. The technique is simple and economical; it allows you to observe the movement of organs and the movement of contrast material in them. However, it also has disadvantages: after it there is no document left that could be discussed or considered in the future. Small image details are difficult to see on the screen. Fluoroscopy is associated with a much greater radiation exposure to the patient and the doctor than radiography.

150. Radiography. In radiography, a beam of x-rays is directed at the part of the body being examined. The radiation passing through the human body hits the film, on which, after processing, an image is obtained.

151. Electroradiography. In it, a beam of X-ray radiation passing through the patient hits a selenium plate charged with static electricity. In this case, the plate changes its electrical potential, and a latent image of electric charges appears on it.

The main advantage of the method is the ability to quickly obtain a large number of high-quality images without consuming X-ray film containing expensive silver compounds and without the “wet” photographic process.

152. Fluorography. Its principle is to photograph an X-ray image from a screen onto a small-format roller film. It is used for mass surveys of the population. The advantages of the method are speed and efficiency.

153. Artificial contrast of organs. The method is based on

introduction into the body of harmless substances that absorb

X-ray radiation is much stronger or, conversely, much weaker than the organ being examined. For example, the patient is recommended to take an aqueous suspension of barium sulfate. In this case, a shadow of a contrast mass located in the stomach cavity appears on the image. By the position, shape, size and outline of the shadow, one can judge the position of the stomach, the shape and size of its cavity.

Iodine is used to contrast the thyroid gland. Gases used for this purpose are oxygen, nitrous oxide, and carbon dioxide. Only nitrous oxide and carbon dioxide can be injected into the bloodstream, since they, unlike oxygen, do not cause gas embolism.

154. X-ray image intensifiers. The brightness of the glow that converts X-ray radiation into visible light of the fluorescent screen, which the radiologist uses when performing fluoroscopy, is hundredths of candelas per square meter (candelas - candle). This roughly corresponds to the brightness of moonlight on a cloudless night. At such illumination, the human eye operates in twilight vision mode, in which small details and weak contrast differences are extremely poorly distinguished.

It is impossible to increase the brightness of the screen due to a proportional increase in the patient’s radiation dose, which is not harmless anyway.

The ability to eliminate this obstacle is provided by X-ray image intensifiers (XI), which are capable of increasing the brightness of images thousands of times by repeatedly accelerating electrons using an external electric field. In addition to increasing brightness, URIs can significantly reduce the radiation dose during research.

155. Angiography– method of contrast study of blood vessels

a system in which, under visual X-ray control using URI and television, a radiologist inserts a thin elastic tube - a catheter - into a vein and directs it along with the blood flow to almost any area of ​​the body, even to the heart. Then, at the right moment, a radiopaque liquid is injected through the catheter and at the same time a series of images is taken, following each other at high speed.

156. Digital method of information processing. Electrical signals are the most convenient form for subsequent image processing. Sometimes it is advantageous to emphasize a line in an image, highlight a contour, or sometimes highlight a texture. Processing can be carried out using both electronic analogue and digital methods. For digital processing purposes, analog signals are converted into discrete form using analog-to-digital converters (ADCs) and are sent to the computer in this form.

The light image obtained on the fluoroscopic screen is amplified by an electron-optical converter (EOC) and enters through the optical system at the input of the TT television tube, turning into a sequence of electrical signals. Using the ADC, sampling and quantization are performed, and then recording into digital random access memory - RAM and processing of image signals according to specified programs. The converted image is again converted into analog form using a DAC digital-to-analog converter and displayed on the screen of the video control device VKU of a grayscale display.

157. Color coding of black and white images. Most introscopic images are monochrome, that is, devoid of color. But normal human vision is color. In order to fully utilize the powers of the eye, it makes sense in some cases to artificially color our introscopic images at the last stage of their transformation.

When the eye perceives color images,

additional image features that facilitate analysis. This

hue, color saturation, color contrast. In color, the visibility of details and the contrast sensitivity of the eye increases many times.

158. X-ray therapy. X-ray radiation is used for radiation therapy in the treatment of a number of diseases. The indications and tactics of radiotherapy are in many ways similar to the methods of gamma therapy.

159. Tomography. The image of an organ or pathological formation of interest to the doctor is overlaid with shadows of neighboring organs and tissues located along the X-ray beam.

The essence of tomography is that during the shooting process

The X-ray tube moves relative to the patient, giving sharp images only of those details that lie at a given depth. Thus, tomography is a layer-by-layer X-ray study.

160. Laser radiation– is a coherent identically directed

radiation from many atoms creating a narrow beam of monochromatic light.

For a laser to start operating, it is necessary to convert a large number of atoms of its working substance into an excited (metastable) state. To do this, electromagnetic energy is transferred to the working substance from a special source (pumping method). After this, almost simultaneous forced transitions of all excited atoms to the normal state will begin in the working substance with the emission of a powerful beam of photons.

161. Application of laser in medicine.High Energy Lasers

used as a laser scalpel in oncology. In this case, rational excision of the tumor is achieved with minimal damage to surrounding tissues, and the operation can be performed near brain structures with great functional significance.

Blood loss when using a laser beam is much less, the wound is completely sterilized, and swelling in the postoperative period is minimal.

Lasers are especially effective in eye microsurgery. It allows the treatment of glaucoma by “piercing” microscopic holes with its beam for the outflow of intraocular fluid. Laser is used for non-surgical treatment of retinal detachment.

Low energy laser radiation has an anti-inflammatory, analgesic effect, changes vascular tone, improves metabolic processes, etc.; it is used in special therapy in various fields of medicine.

162. Effect of laser on the body. The impact of laser radiation on the body is in many ways similar to the impact of electromagnetic radiation in the visible and infrared ranges. At the molecular level, such an effect leads to a change in the energy levels of molecules of living matter, their stereochemical rearrangement, and coagulation of protein structures. The physiological effects of laser exposure are associated with the photodynamic effect of photoreactivation, the effect of stimulation or inhibition of biological processes, changes in the functional state of both individual systems and the body as a whole.

163. Use of lasers in biomedical research. One of the main areas of laser diagnostics is condensed matter spectroscopy, which allows for the analysis of biological tissues and their visualization at the cellular, subcellular and molecular levels.

where l is the distance between the upper focus of the lens and the lower focus of the eyepiece; L – distance of best vision; equal to 25 cm; F 1 and F 2 – focal lengths of the lens and eyepiece.

Knowing the focal lengths F 1, F 2 and the distance between them l, you can find the magnification of the microscope.

In practice, microscopes with magnifications greater than 1500–2000 are not used, because The ability to distinguish small details of an object in a microscope is limited. This limitation is caused by the influence of light diffraction in the passing structure of a given object. In this regard, the concepts of resolution limit and resolving power of a microscope are used.

Determining the limit of microscope resolution

Microscope resolution limit is the smallest distance between two points on an object at which they are visible separately in a microscope. This distance is determined by the formula:

where λ is the wavelength of light; n is the refractive index of the medium between the lens and the object; u is the aperture angle of the lens, equal to the angle between the outer rays of the conical light beam entering the microscope lens.

In reality, light from an object propagates to the microscope lens in a certain cone (Fig. 2 a), which is characterized by an angular aperture - the angle u between the outer rays of a conical light beam entering the optical system. In the limiting case, according to Abbe, the outer rays of the conical light beam will be the rays corresponding to the central (zero) and 1st main maxima (Fig. 2 b).

The quantity 2nsin U is called the numerical aperture of the microscope. The numerical aperture can be increased using a special liquid medium - immersion– in the space between the objective and the cover glass of the microscope.

In immersion systems, compared to identical “dry” systems, a larger aperture angle is obtained (Fig. 3).

Fig.3. Immersion system diagram

Water (n = 1.33), cedar oil (n = 1.514), etc. are used as immersion. For each immersion, a lens is specially calculated, and it can only be used with this immersion.

The formula shows that the resolution limit of the microscope depends on the wavelength of light and the numerical aperture of the microscope. The shorter the wavelength of light and the larger the aperture, the smaller Z, and, therefore, the greater the resolution limit of the microscope. For white (daylight) light, the average wavelength can be taken as λ = 0.55 µm. The refractive index for air is n = 1.

Microscope mbs-1

MBS-1 is a stereoscopic microscope that provides a direct three-dimensional image of the object under consideration in both transmitted and reflected light.

The microscope consists of 4 main parts:

– table;

– tripod;

– optical head with a coarse feed mechanism;

– eyepiece attachment.

The microscope stage consists of a round body, inside of which a rotating reflector with mirror and matte surfaces is mounted. To work with daylight, the housing has a cutout through which light passes freely. On the back side of the table body there is a threaded hole for working with an electric illuminator. An optical head is attached to the microscope stand - the main part of the device, into which the most important optical components are mounted.

The housing of the optical head contains a drum with Galilean systems installed in it. Rotate the drum axis using handles with printed numbers 0.6; 1; 2; 4; 7 achieve different lens magnifications. Each position of the drum is clearly fixed with a special spring clamp. Using the handle on the microscope tripod, which moves the optical head, the sharpest image of the object in question is achieved.

The entire optical head can be moved on the tripod rod and secured in any position with a screw. The eyepiece attachment consists of a guide, which is a rectangular piece with two holes for lens frames.

When observing through the eyepieces, you need to turn the eyepiece tubes to find a position in which the two images are combined into one. Next, focus the microscope on the object under study, and rotate the reflector to achieve uniform illumination of the field. When adjusting the illumination, the socket with the lamp moves towards the collector until the best illumination of the observed object is obtained.

Basically, MBS-1 is intended for preparation work, for observing objects, as well as for carrying out linear measurements or measuring the areas of sections of the preparation. The optical diagram of the microscope is shown in Fig. 4.

The optical diagram of the MBS-1 microscope is shown in Fig. 4.

When working in transmitted light, the light source (1) with the help of a reflector (2) and a collector (3) illuminates a transparent specimen mounted on the stage (4).

A special system was used as a lens, consisting of 4 lenses (5) with a focal length = 80 mm and 2 pairs of Galilean systems (6) and (7), behind which there are lenses (8) with a focal length of 160 mm, which form an image of the object in the focal planes of the eyepieces.

The total linear magnification of the optical system, consisting of a lens (5), Galilean systems (6) and (7) and lenses (8) is: 0.6; 1; 2; 4; 7. Behind the lenses (8) there are 2 Schmidt prisms (9), which allow you to rotate the eyepiece tubes according to the observer’s eye without rotating the lens image.

The MBS-1 microscope comes with 3 pairs of eyepieces (10) with a magnification of 6; 8; 12.5 and one 8x magnification eyepiece micrometer with reticle. They allow you to vary the overall magnification of the microscope from 3.6 to 88 (Table 1). The total magnification of a microscope is the product of the magnification of the eyepiece and the magnification of the objective.

Table 1.

Optical characteristics of the MBS-1 microscope

2. Optical system of the microscope.

3. Microscope magnification.

4. Resolution limit. Resolution power of the microscope.

5. Useful microscope magnification.

6. Special techniques of microscopy.

7. Basic concepts and formulas.

8. Tasks.

The ability of the eye to distinguish small 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 = 1-10 cm).

The object in question is placed between the magnifying glass and its front focus in such a way that its virtual image is within the limits of accommodation for a given eye. Usually the 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 viewing angles at which an object is visible when viewed “nakedly” normal with the eye and with a magnifying glass. We will perform the calculations for the case when a virtual image of an object is obtained at infinity (the far limit of accommodation).

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

When viewing 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 the object B", located in an infinitely distant plane. The viewing angle at which the image is visible is equal to β" ≈ B/f.

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

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 an object is visible to the “naked” normal eye from the distance of best vision:

Magnification magnifications are different for nearsighted and farsighted eyes, since they have different distances of best vision.

Let us present without derivation the formula for the magnification given by a magnifying glass used by a nearsighted or farsighted eye when forming an image in the plane of far accommodation:

where a distance is the far limit of accommodation.

Formula (25.1) suggests that by reducing the focal length of the magnifying glass, you can achieve an arbitrarily large magnification. In principle this is true. However, when the focal length of a magnifying glass is reduced and its size remains the same, aberrations arise that negate the entire effect of magnification. Therefore, single-lens magnifiers usually have 5-7x magnification.

To reduce aberrations, complex magnifying glasses are made, consisting of two or three lenses. In this case, it is possible to achieve a 50-fold increase.

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. In this case the magnifying glass is called eyepiece, and the other lens - lens. The path of rays in a microscope is shown in Fig. 25.2.

Object B is placed near the front focus of the lens (F about) in such a way that its actual, magnified image B" is located between the eyepiece and its front focus. When

Rice. 25.2. Path of rays in a microscope.

In this case, the eyepiece gives an imaginary magnified image B", which is viewed by the eye.

By changing the distance between the object and the lens, they ensure that the image B" is in the plane of far accommodation of the eye (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 a microscope is its angular increase. This concept is similar 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 best vision (a 0):

25.4. Resolution limit. Microscope resolution

You may get the impression that by increasing the optical length of the tube, you can achieve an arbitrarily large magnification and, therefore, examine the smallest details of an object.

However, taking into account the wave properties of light shows that the size of small details discernible using a microscope is subject to restrictions associated with diffraction light passing through the lens opening. Due to diffraction, the image of an illuminated point is not a point, but small light circle. If the parts (points) of the object under consideration are located far enough away, 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 located very close, then the corresponding “circles” overlap and are perceived as one object (Fig. 25.3, c).

Rice. 25.3. Resolution

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

Resolution limit microscope (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 reciprocal of the resolution limit is called resolution. The lower the resolution limit, the greater the resolution.

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

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

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

Where λ - the wavelength of light that illuminates the object.

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

25.5. Useful microscope magnification

Let's find out how large the microscope's magnification should be for a given resolution limit of its lens. Let us take into account that the eye has its own resolution limit, determined by the structure of the retina. In Lecture 24 we obtained the following estimate for eye resolution limit: ZGL = 145-290 µm. In order for the eye to distinguish the same points that are separated by a microscope, magnification is necessary.

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 whose size is equal to the resolution limit of the eye.

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

It makes no sense to achieve a higher magnification value for the microscope, since no additional details will be visible anyway.

Useful microscope magnification - it is a reasonable combination of the resolving powers of both the microscope and the eye.

25.6. Special Microscopy Techniques

Special microscopy techniques are used to increase the resolving power (decreasing the resolution limit) of the microscope.

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

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

2. UV microscopy. For decreasing resolution limit They use short-wave ultraviolet radiation, invisible to the eye. In ultraviolet microscopes, a microobject 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 fluorescent screen).

3. Measuring the size of microscopic objects. Using a microscope, you can determine the size of the observed object. An eyepiece micrometer is used for this. The simplest eyepiece micrometer is a round glass plate on which a graduated scale is applied. The micrometer is installed in the plane of the image obtained from the lens. When viewed through the eyepiece, the images of the object and the scale merge, and you can calculate which distance on the scale corresponds to the measured value. The division price of the ocular micrometer is preliminarily determined from a known object.

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

5. Ultramicroscopy. The microscope can detect particles whose sizes lie beyond its resolution. This method uses oblique illumination, due to which microparticles are visible as light dots on a dark background, while the structure of the particles cannot be seen, the fact of their presence can only be established.

The theory shows that, no matter how powerful the microscope is, any object smaller than 3 microns will be represented in it simply as one point, without any details. But this does not mean that such particles cannot be seen, their movements can be monitored, or they cannot be counted.

To observe particles whose sizes are smaller than the resolution limit of the microscope, a device called ultramicroscope. The main part of the ultramicroscope is a strong lighting 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 (think of 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 length of the tube is 18 cm. What is the magnification of the microscope?

2. Determine the resolution limit of dry and immersion (n = 1.55) lenses with an angular aperture u = 140 o. Take the wavelength to be 0.555 µm.

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

4. What refractive index should an immersion liquid be used 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 aperture angle of the microscope is 70°.

5. There is an inscription “x10” on the rim of the magnifying glass. Determine the focal length of this magnifying glass.

6. Microscope lens focal length f 1 = 0.3 cm, tube length Δ = 15 cm, magnification Г = 2500. Find the focal length F 2 of the eyepiece. The best vision distance is a 0 = 25 cm.

The resolution of the eye is limited. Resolution characterized resolved distance, i.e. the minimum distance between two neighboring particles at which they are still visible separately. The resolved distance for the naked eye is about 0.2 mm. A microscope is used to increase resolution. To study the structure of metals, the microscope was first used in 1831 by P.P. Anosov, who studied damask steel, and later, in 1863, by the Englishman G. Sorby, who studied meteorite iron.

The permitted distance is determined by the relationship:

Where l- wavelength of light coming from the object of study to the lens, n– refractive index of the medium located between the object and the lens, and a- angular aperture equal to half the opening angle of the beam of rays entering the lens that produces the image. This important characteristic of the lens is engraved on the lens frame.

Good lenses have a maximum aperture angle a = 70° and sina » 0.94. Most studies use dry objectives operating in air (n = 1). To reduce the resolved distance, immersion lenses are used. The space between the object and the lens is filled with a transparent liquid (immersion) with a high refractive index. Typically a drop of cedar oil is used (n = 1.51).

If we take l = 0.55 µm for visible white light, then the minimum resolving distance of a light microscope is:

Thus, the resolving power of a light microscope is limited by the wavelength of light. The lens magnifies the intermediate image of the object, which is viewed through the eyepiece, as if through a magnifying glass. The eyepiece magnifies the intermediate image of the object and cannot increase the resolution of the microscope.

The total magnification of the microscope is equal to the product of the magnification of the objective and the eyepiece. Metallographic microscopes are used to study the structure of metals with magnification from 20 to 2000 times.

Beginners make a common mistake by trying to view the structure immediately at high magnification. It should be kept in mind that the greater the magnification of an object, the smaller the area visible in the field of view of the microscope. Therefore, it is recommended to begin the study by using a weak lens in order to first assess the general nature of the metal structure over a large area. If you start microanalysis using a strong lens, then many important features of the metal structure may not be noticed.

After a general view of the structure at low magnifications of the microscope, a lens with such a resolution is selected to see all the necessary smallest details of the structure.

The eyepiece is chosen so that the details of the structure, magnified by the lens, are clearly visible. If the eyepiece magnification is not sufficient, the fine details of the intermediate image created by the lens will not be seen through the microscope, and thus the full resolution of the lens will not be used. If the eyepiece magnification is too high, new structural details will not be revealed, at the same time, the contours of already identified details will be blurred, and the field of view will become narrower. The eyepiece's own magnification is engraved on its frame (for example, 7 x).