On what values does the resolution of a microscope depend? Resolution and magnification of the microscope. Microscope optical system
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).
A microscope is designed to observe small objects with greater magnification and greater resolution than a magnifying glass provides. The optical system of a microscope consists of two parts: a lens and an eyepiece. The microscope lens forms a true magnified inverse image of the object in the front focal plane of the eyepiece. The eyepiece acts like a magnifying glass and forms a virtual image at the best viewing distance. In relation to the entire microscope, the object in question is located in the front focal plane.
Microscope Magnification
The action of a microlens is characterized by its linear magnification: V ob = -Δ/F\" ob * F\" ob - focal length of the microlens * Δ - distance between the rear focus of the lens and the front focus of the eyepiece, called the optical interval or optical length of the tube.
The image created by the microscope objective at the front focal plane of the eyepiece is viewed through the eyepiece, which acts as a magnifying glass with visible magnification:
G ok =¼ F ok
The overall magnification of a microscope is determined as the product of the objective magnification and the eyepiece magnification: G=V about *G approx
If the focal length of the entire microscope is known, then its apparent magnification can be determined in the same way as for a magnifying glass:
As a rule, the magnification of modern microscope lenses is standardized and amounts to a series of numbers: 10, 20, 40, 60, 90, 100 times. Eyepiece magnifications also have very specific values, for example 10, 20, 30 times. All modern microscopes have a set of objectives and eyepieces that are specially designed and manufactured to fit together so that they can be combined to achieve different magnifications.
Field of view of the microscope
The field of view of the microscope depends on the angular field of the eyepiece ω , within which an image of fairly good quality is obtained: 2y=500*tg(ω)/G * G - microscope magnification
For a given angular field of the eyepiece, the linear field of the microscope in object space is smaller, the greater its apparent magnification.
Microscope exit pupil diameter
The exit pupil diameter of a microscope is calculated as follows:
where A is the front aperture of the microscope.
The diameter of the exit pupil of a microscope is usually slightly smaller than the diameter of the pupil of the eye (0.5 - 1 mm).
When observing through a microscope, the pupil of the eye must be aligned with the exit pupil of the microscope.
Microscope resolution
One of the most important characteristics of a microscope is its resolution. According to Abbe's diffraction theory, the linear resolution limit of a microscope, that is, the minimum distance between points on an object that are imaged as separate, depends on the wavelength and numerical aperture of the microscope:
The maximum achievable resolution of an optical microscope can be calculated based on the expression for the microscope aperture. If we take into account that the maximum possible value of the sine of the angle is unity, then for the average wavelength we can calculate the resolution of the microscope: 
There are two ways to increase the resolution of a microscope: * By increasing the objective aperture, * By decreasing the wavelength of light.
Immersion
In order to increase the aperture of the lens, the space between the object in question and the lens is filled with the so-called immersion liquid - a transparent substance with a refractive index greater than one. Water, cedar oil, glycerin solution and other substances are used as such a liquid. The apertures of high-magnification immersion objectives reach the value , then the maximum achievable resolution of an immersion optical microscope will be. 
Application of ultraviolet rays
To increase the resolution of a microscope, the second method uses ultraviolet rays, the wavelength of which is shorter than that of visible rays. In this case, special optics must be used that are transparent to ultraviolet light. Since the human eye does not perceive ultraviolet radiation, it is necessary either to resort to means that convert the invisible ultraviolet image into a visible one, or to photograph the image in ultraviolet rays. At the wavelength, the resolution of the microscope will be.
In addition to increased resolution, the ultraviolet light observation method has other advantages. Typically, living objects are transparent in the visible region of the spectrum, and therefore are pre-stained before observation. But some objects (nucleic acids, proteins) have selective absorption in the ultraviolet region of the spectrum, due to which they can be “visible” in ultraviolet light without staining.
Image quality determined microscope resolution, i.e. the minimum distance at which the optics of a microscope can separately distinguish two closely spaced points. resolution depends on the numerical aperture of the objective, condenser and the wavelength of light with which the specimen is illuminated. The numerical aperture (opening) depends on the angular aperture and refractive index of the medium located between the front lens of the objective and condenser and the specimen.
Lens Angular Aperture- this is the maximum angle (AOB) at which rays passing through the preparation can enter the lens. Lens Numerical Aperture equal to the product of the sine of half the angular aperture and the refractive index of the medium located between the glass slide and the front lens of the objective lens. N.A. = n sinα where, N.A. - numerical aperture; n is the refractive index of the medium between the specimen and the lens; sinα is the sine of angle α equal to half the angle AOB in the diagram.
Thus, the aperture of dry systems (between the front objective lens and the air preparation) cannot be more than 1 (usually no more than 0.95). The medium placed between the specimen and the objective is called immersion liquid or immersion, and an objective designed to work with immersion liquid is called immersion. Thanks to immersion with a higher refractive index than air, it is possible to increase the numerical aperture of the lens and, therefore, the resolution.
The numerical aperture of lenses is always engraved on their frames.
The resolution of the microscope also depends on the aperture of the condenser. If we consider the condenser aperture to be equal to the lens aperture, then the resolution formula has the form R=λ/2NA, where R is the resolution limit; λ - wavelength; N.A - numerical aperture. From this formula it is clear that when observed in visible light (green part of the spectrum - λ = 550 nm), the resolution (resolution limit) cannot be > 0.2 µm
The influence of the numerical aperture of the microscope objective on image quality
Ways to increase optical resolution
Selecting a large light cone angle, both from the lens side and from the light source side. Thanks to this, it is possible to collect more refracted rays of light from very thin structures in the lens. Thus, the first way to increase resolution is to use a condenser whose numerical aperture matches the numerical aperture of the objective.
The second method is to use immersion liquid between the front objective lens and the cover glass. This is how we influence the refractive index of the medium n, described in the first formula. Its optimal value recommended for immersion liquids is 1.51.
Immersion liquids
Immersion liquids are necessary to increase the numerical aperture and, accordingly, increase the resolution of immersion objectives, specially designed to work with these liquids and, accordingly, marked. Immersion liquids placed between the objective and the specimen have a higher refractive index than air. Therefore, light rays deflected by the smallest details of the object are not scattered when leaving the preparation and enter the lens, which leads to an increase in resolution.
There are water immersion lenses (marked with a white ring), oil immersion lenses (black ring), glycerin immersion lenses (yellow ring), and monobromonaphthalene immersion lenses (red ring). In light microscopy of biological preparations, water and oil immersion objectives are used. Special quartz glycerol immersion objectives transmit short-wave ultraviolet radiation and are designed for ultraviolet (not to be confused with fluorescent) microscopy (that is, for studying biological objects that selectively absorb ultraviolet rays). Monobrominated naphthalene immersion objectives are not used in the microscopy of biological objects.
Distilled water is used as an immersion liquid for a water immersion lens, and natural (cedar) or synthetic oil with a certain refractive index is used as an immersion liquid for an oil immersion lens.
Unlike other immersion liquids oil immersion is homogeneous because it has a refractive index equal to or very close to the refractive index of glass. Typically this refractive index (n) is calculated for a specific spectral line and a specific temperature and is indicated on the oil bottle. For example, the refractive index of immersion oil for working with a cover glass for spectral line D in the sodium spectrum at a temperature = 20°C is 1.515 (nD 20 = 1.515), for working without a cover glass (nD 20 = 1.520).
To work with apochromatic lenses, dispersion is also normalized, that is, the difference in refractive indices for different lines of the spectrum.
The use of synthetic immersion oil is preferable because its parameters are more accurately standardized, and unlike cedar oil, it does not dry out on the surface of the front lens of the lens.
Considering the above, in no case should you use surrogates for immersion oil and, in particular, vaseline oil. In some microscopy methods, to increase the aperture of the condenser, an immersion liquid (usually distilled water) is placed between the condenser and the specimen.
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 that differ from the environment only in 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, falls 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 have any external impact or inconvenience for 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 both 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 similar 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 counterfeit drugs. 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 is 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 beam. 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 electrical 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.
Light microscopy
Light microscopy provides magnification up to 2-3 thousand times, a color and moving image of a living object, the possibility of micro-filming and long-term observation of the same object, assessment of its dynamics and chemistry.
The main characteristics of any microscope are resolution and contrast. Resolution is the minimum distance at which two points are located, demonstrated separately by the microscope. The resolution of the human eye in the best vision mode is 0.2 mm.
Image contrast is the difference in brightness between the image and the background. If this difference is less than 3 - 4%, then it cannot be caught either by the eye or by a photographic plate; then the image will remain invisible, even if the microscope resolves its details. Contrast is influenced both by the properties of the object, which change the luminous flux compared to the background, and by the ability of the optics to capture the resulting differences in the properties of the beam.
The capabilities of a light microscope are limited by the wave nature of light. The physical properties of light - color (wavelength), brightness (wave amplitude), phase, density and direction of wave propagation change depending on the properties of the object. These differences are used in modern microscopes to create contrast.
The magnification of a microscope is defined as the product of the objective magnification and the magnification of the eyepiece. Typical research microscopes have an eyepiece magnification of 10, and an objective magnification of 10, 45 and 100. Accordingly, the magnification of such a microscope ranges from 100 to 1000. Some microscopes have a magnification of up to 2000. Even higher magnification does not make sense, since resolution does not improve. On the contrary, the image quality deteriorates.
Numerical aperture is used to express the resolving power of an optical system or the aperture ratio of a lens. Lens aperture is the light intensity per unit area of the image, approximately equal to the square of NA. The NA value is approximately 0.95 for a good lens. The microscope is usually sized so that its total magnification is about 1000 NA. If a liquid (oil or, more rarely, distilled water) is introduced between the objective and the sample, an “immersion” objective is obtained with an NA value as high as 1.4 and a corresponding improvement in resolution.
Light microscopy methods
Light microscopy methods (illumination and observation). Microscopy methods are selected (and provided constructively) depending on the nature and properties of the objects being studied, since the latter, as noted above, affect the image contrast.
Bright field method and its varieties
The bright field method in transmitted light is used to study transparent preparations with absorbing (light-absorbing) particles and parts included in them. These can be, for example, thin colored sections of animal and plant tissues, thin sections of minerals, etc. In the absence of a preparation, a beam of light from the condenser, passing through the lens, produces a uniformly illuminated field near the focal plane of the eyepiece. If there is an absorbent element in the preparation, partial absorption and partial scattering of the light incident on it occurs, which causes the appearance of the image. It is also possible to use the method when observing non-absorbing objects, but only if they scatter the illuminating beam so strongly that a significant part of it does not fall into the lens.
The oblique lighting method is a variation of the previous method. The difference between them is that the light is directed at the object at a large angle to the direction of observation. Sometimes this helps to reveal the “relief” of an object due to the formation of shadows.
The bright field method in reflected light is used when studying opaque objects that reflect light, such as polished sections of metals or ores. The preparation is illuminated (from an illuminator and a translucent mirror) from above, through a lens, which simultaneously plays the role of a condenser. In the image created in a plane by the lens together with the tube lens, the structure of the preparation is visible due to the difference in the reflectivity of its elements; In the bright field, inhomogeneities that scatter the light incident on them also stand out.
Dark field method and its variations
The dark-field microscopy method is used to obtain images of transparent, non-absorbent objects that cannot be seen using the bright-field method. Often these are biological objects. Light from the illuminator and mirror is directed onto the preparation by a specially designed condenser - the so-called. dark field condenser. Upon exiting the condenser, the main part of the light rays, which did not change their direction when passing through the transparent preparation, forms a beam in the form of a hollow cone and does not enter the lens (which is located inside this cone). The image in the microscope is formed using only a small part of the rays scattered by microparticles of the drug located on the slide into the cone and passing through the 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. In the field of view against a dark background, light images of the structural elements of the drug are visible, which differ from the surrounding environment in their refractive index. Large particles have only bright edges that scatter light rays. Using this method, it is impossible to determine from the appearance of the image whether the particles are transparent or opaque, or whether they have a higher or lower refractive index compared to the surrounding medium.
Conducting a dark-field study
Slides should be no thicker than 1.1-1.2 mm, coverslips 0.17 mm, without scratches or dirt. When preparing the drug, you should avoid the presence of bubbles and large particles (these defects will be visible with a bright glow and will not allow you to observe the drug). For dark-field, more powerful illuminators and maximum lamp intensity are used.
Setting up darkfield lighting is basically as follows:
Install the light according to Koehler;
Replace the bright-field condenser with a dark-field one;
Immersion oil or distilled water is applied to the upper condenser lens;
Raise the condenser until it touches the bottom surface of the slide;
A low magnification lens is focused on the specimen;
Using centering screws, a light spot (sometimes having a darkened central area) is transferred to the center of the field of view;
By raising and lowering the condenser, the darkened central area disappears and a uniformly illuminated light spot is obtained.
If this cannot be done, then you need to check the thickness of the glass slide (this phenomenon is usually observed when using too thick glass slides - the cone of light is focused in the thickness of the glass).
After setting the light correctly, install a lens of the required magnification and examine the specimen.
The ultramicroscopy method is based on the same principle - preparations in ultramicroscopes are illuminated perpendicular to the direction of observation. With this method, it is possible to detect (but not literally “observe”) extremely small particles, the sizes of which lie far beyond the resolution of the most powerful microscopes. With the help of immersion ultramicroscopes, it is possible to register the presence in a preparation of particles × particles up to 2 × 10 to -9 degrees m in size. But the shape and exact dimensions of such particles cannot be determined using this method. Their images appear to the observer in the form of diffraction spots, the dimensions of which depend not on the size and shape of the particles themselves, but on the aperture of the lens and the magnification of the microscope. Since such particles scatter very little light, extremely strong light sources, such as a carbon electric arc, are required to illuminate them. Ultramicroscopes are used mainly in colloid chemistry.
Phase contrast method
The phase contrast method and its variety - the so-called. The “anoptral” contrast method is designed to obtain images of transparent and colorless objects that are invisible when observed using the bright field method. These include, for example, living undyed animal tissues. The essence of the method is that even with very small differences in the refractive indices of different elements of the preparation, the light wave passing through them undergoes different changes in phase (acquires the so-called phase relief). Not perceived directly by either the eye or the photographic plate, these phase changes are converted with the help of a special optical device into changes in the amplitude of the light wave, i.e., into changes in brightness (“amplitude relief”), which are already visible to the eye or recorded on the photosensitive layer. In other words, in the resulting visible image, the distribution of brightness (amplitude) reproduces the phase relief. The image obtained in this way is called phase-contrast.
The phase contrast device can be installed on any light microscope and consists of:
A set of lenses with special phase plates;
Condenser with rotating disk. It contains annular diaphragms corresponding to the phase plates in each of the lenses;
An auxiliary telescope for adjusting phase contrast.
The phase contrast setting is as follows:
Replace the lenses and condenser of the microscope with phase ones (indicated by the letters Ph);
Install a low magnification lens. The hole in the condenser disk must be without an annular diaphragm (indicated by the number "0");
Adjust the light according to Koehler;
Select a phase lens of appropriate magnification and focus it on the specimen;
Turn the condenser disk and install the annular diaphragm corresponding to the lens;