Polarized microscopy. Visualization of intracellular structures of microorganisms using light microscopy. Phase contrast microscopy method
![Polarized microscopy. Visualization of intracellular structures of microorganisms using light microscopy. Phase contrast microscopy method](https://i0.wp.com/meduniver.com/Medical/Microbiology/Img/211.jpg)
Polarization microscopy allows you to obtain images of unstained anisotropic structures (for example, collagen fibers, myofibrils or microbial cells). The principle of the method is based on studying an object in light formed by two beams polarized in mutually perpendicular planes.
Rice. 11-4. Diagram of a fluorescent microscope.Interference microscopy
Interference microscopy combines the principles of phase-contrast and polarization microscopy. The method is used to obtain a contrasting three-dimensional image of unpainted objects. The principle of the method is based on splitting the light flux in a microscope; one ray passes through the object, the other - past it. Both beams are connected at the eyepiece and interfere with each other.
![](https://i0.wp.com/meduniver.com/Medical/Microbiology/Img/211.jpg)
Fluorescence microscopy
Fluorescence microscopy method based on the ability of some substances to glow when exposed to short-wave radiation. In this case, the emitted light waves are longer than the wave that causes the glow. In other words, fluorescent objects absorb light of one wavelength and emit light in another region of the spectrum (Fig. 11-4). For example, if the inducing radiation is blue, then the resulting glow may be red or yellow. These substances (fluorescein isocyanate, acridine orange, rhodamine, etc.) are used as fluorescent dyes for observing fluorescent (luminescent) objects. In a fluorescence microscope, light from a source (ultra-high pressure mercury lamp) passes through two filters.
![](https://i2.wp.com/meduniver.com/Medical/Microbiology/Img/212.jpg)
First (blue) filter traps light in front of the sample and transmits light of the wavelength that excites fluorescence of the sample. The second (yellow) blocks blue light, but transmits yellow, red, green light emitted by a fluorescent object and perceived by the eye. Typically, the microorganisms under study are stained directly or using AT or lectins labeled with fluorochromes. The drugs interact with Ag or other ligand-binding structures of the object. Fluorescence microscopy has found wide application for visualizing the results of immunochemical reactions based on the specific interaction of AT labeled with fluorescent dyes with Ag of the studied object. Options immunofluorescent reactions are presented in Fig. 11-5 and 11-6.
Send your good work in the knowledge base is simple. Use the form below
Students, graduate students, young scientists who use the knowledge base in their studies and work will be very grateful to you.
Posted on http://www.allbest.ru/
Introduction
Light microscopy
Electron microscopy
Polarization microscopy
Annex 1
Light microscopy
Light microscopy is the most ancient and at the same time one of the most common methods for studying and studying plant and animal cells. It is assumed that the beginning of the study of cells was precisely with the invention of the light optical microscope. The main characteristic of a light microscope is the resolution of the light microscope, which is determined by the wavelength of the light. The resolution limit of a light microscope is determined by the wavelength of light; an optical microscope is used to study structures that have minimal dimensions equal to the wavelength of light radiation. Many constituent cells are similar in optical density and require pre-treatment before microcopying, otherwise they are practically invisible under a conventional light microscope. In order to make them visible, various dyes with a certain selectivity are used. Using selective dyes, it becomes possible to study the internal structure of the cell in more detail.
For example:
hematoxylin dye colors some components of the nucleus blue or violet;
after treatment sequentially with phloroglucinol and then with hydrochloric acid, the lignified cell membranes become cherry red;
Sudan III dye stains suberized cell membranes pink;
a weak solution of iodine in potassium iodide turns starch grains blue.”
When conducting microscopic examinations, most tissues are fixed before staining.
Once fixed, the cells become permeable to dyes and the cell structure is stabilized. One of the most common fixatives in botany is ethyl alcohol.
During the preparation of the preparation for microcopying, thin sections are made on a microtome (Appendix 1, Fig. 1). This device uses the bread slicer principle. Slightly thicker sections are made for plant tissues than for animal tissues because plant cells are relatively larger. Thickness of plant tissue sections for - 10 microns - 20 microns. Some tissues are too soft to cut straight away. Therefore, after fixation, they are poured into molten paraffin or special resin, which saturates the entire fabric. After cooling, a solid block is formed, which is then cut using a microtome. This is explained by the fact that plant cells have strong cell walls that make up the tissue frame. Lignified shells are especially durable.
When using the filling during preparation, the cut runs the risk of damaging the cell structure; to prevent this, use the method of quick freezing. When using this method, you can do without fixing and filling. Frozen tissue is cut using a special microtome - cryotome (Appendix 1, Fig. 2).
Frozen sections better preserve natural structural features. However, they are more difficult to cook and the presence of ice crystals ruins some of the details.
phase-contrast (Appendix 1, Fig. 3) and interference microscopes (Appendix 1, Fig. 4) allow you to examine living cells under a microscope with a clear manifestation of the details of their structure. These microscopes use 2 beams of light waves that interact (superpose) on each other, increasing or decreasing the amplitude of the waves entering the eye from different components of the cell.
Light microscopy has several varieties.
Bright field method and its varieties
Transmitted light field method used in the study of transparent preparations containing light-absorbing particles and details (thin colored sections of animal and plant tissues, thin sections of minerals). In the absence of the drug, 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.
Oblique lighting method- 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.
Bright field method in reflected light used when studying opaque light-reflecting objects, such as thin 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
Transmitted light dark field method 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. 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.
Electron microscopy
The first electron microscope was constructed in 1931 by Knoll and Ruska in Germany. It was only in the 50s that methods for producing sections with the necessary qualities were developed.
The difficulties of electron microscopy are that special processing of preparations is necessary to study biological samples.
The first difficulty is that electrons have very limited penetrating power, so ultrathin sections, 50 - 100 nm thick, must be prepared. To obtain such thin sections, the tissue is first impregnated with resin: the resin polymerizes to form a hard plastic block. Then, using a sharp glass or diamond knife, the sections are cut on a special microtome.
There is another difficulty: when electrons pass through biological tissue, a contrast image is not obtained. In order to obtain contrast, thin sections of biological samples are impregnated with salts of heavy metals.
There are two main types of electron microscopes. In a transmission (transmission) microscope, a beam of electrons, passing through a specially prepared sample, leaves its image on the screen. The resolution of a modern transmission electron microscope is almost 400 times greater than that of light. These microscopes have a resolution of about 0.5 nm.
Despite such high resolution, transmission electron microscopes have major disadvantages:
you have to work with fixed materials;
the image on the screen is two-dimensional (flat);
When treated with heavy metals, some cellular structures are destroyed and modified.
A three-dimensional (volumetric) image is obtained using a scanning electron microscope (EM). Here the beam does not pass through the sample, but is reflected from its surface.
The test sample is fixed and dried, after which it is covered with a thin layer of metal, an operation called shading (the sample is shaded).
In scanning EM, a focused electron beam is directed onto a sample (the sample is scanned). As a result, the metal surface of the sample emits secondary electrons of low energy. They are recorded and converted into an image on a television screen. The maximum resolution of a scanning microscope is small, about 10 nm, but the image is three-dimensional.
Types of electron microscopy:
Amplitude electron microscopy- Methods of amplitude electron microscopy can be used to process images of amorphous and other bodies (the particle sizes of which are smaller than the distance resolved in an electron microscope) that scatter electrons diffusely. In a transmission electron microscope, for example, the contrast of the image, i.e., the difference in brightness of the image of neighboring areas of the object, is, to a first approximation, proportional to the difference in the thickness of these areas.
Phase electron microscopy- To calculate the contrast of images of crystalline bodies with regular structures, as well as to solve the inverse problem - calculating the structure of an object from an observed image - phase electron microscopy methods are used. The problem of diffraction of an electron wave on a crystal lattice is considered, the solution of which additionally takes into account the inelastic interactions of electrons with an object: scattering by plasmas, phonons, etc. In transmission electron microscopes and high-resolution scanning transmission electron microscopes, images of individual molecules or atoms of heavy elements are obtained . Using phase electron microscopy methods, it is possible to reconstruct the three-dimensional structure of crystals and biological macromolecules from images.
Quantitative electron microscopy- Quantitative electron microscopy methods are the precise measurement of various parameters of a sample or process under study, for example, measurement of local electric potentials, magnetic fields, microgeometry of surface relief, etc.
Lorentz electron microscopy- The field of study of Lorentz electron microscopy, in which phenomena caused by the Lorentz force are studied, are internal magnetic and electric fields or external stray fields, for example, the fields of magnetic domains in thin films, ferroelectric domains, fields of heads for magnetic recording of information, etc.
Polarization microscopy
Polarization microscopy is an observation method in polarized light for the microscopic examination of preparations containing optically anisotropic elements (or consisting entirely of such elements). These include many minerals, grains in thin sections of alloys, some animal and plant tissues, etc. Observation can be carried out both in transmitted and reflected light. The light emitted by the illuminator is passed through a polarizer. The polarization imparted to it changes with the subsequent passage of light through the preparation (or reflection from it). These changes are studied using an analyzer and various optical compensators. By analyzing such changes, one can judge the main optical characteristics of anisotropic microobjects: the strength of birefringence, the number of optical axes and their orientation, rotation of the plane of polarization, and dichroism.
Phase contrast method
Method phase contrast and its variety - the so-called. method "anoptral" contrast are 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.
A typical operating diagram of the method: an aperture diaphragm is installed in the front focus of the condenser, the hole of which has the shape of a ring. Its image appears near the rear focus of the lens, and the so-called. a phase plate on the surface of which there is an annular protrusion or annular groove, called a phase ring. The phase plate is not always placed at the focus of the lens - often the phase ring is applied directly to the surface of one of the objective lenses.
In any case, the rays from the illuminator that are not deflected in the preparation, giving an image of the diaphragm, must completely pass through the phase ring, which significantly weakens them (it is made absorbing) and changes their phase by l/4 (l is the wavelength of light). And the rays, even slightly deflected (scattered) in the preparation, pass through the phase plate, bypassing the phase ring, and do not undergo an additional phase shift.
Taking into account the phase shift in the preparation material, the total phase difference between the deflected and non-deviated beams is close to 0 or l/2, and as a result of the interference of light in the image plane of the preparation, they noticeably enhance or weaken each other, giving a contrasting image of the structure of the preparation. Deflected rays have a significantly lower amplitude compared to non-deviated ones, therefore, weakening the main beam in the phase ring, bringing the amplitude values closer together, also leads to greater image contrast.
The method makes it possible to distinguish small structural elements that are extremely low-contrast in the bright field method. Transparent particles, which are relatively small in size, scatter light rays at such small angles that these rays pass together with those not deflected through the phase ring. For such particles, the phase-contrast effect occurs only near their contours, where strong scattering occurs.
Infrared observation method
Method observations in infrared(IR) rays also require converting an image invisible to the eye into a visible one using photography or using an electron-optical converter. IR microscopy makes it possible to study the internal structure of objects that are opaque in visible light, such as dark glasses, some crystals and minerals, etc.
Ultraviolet Observation Method
Method observations in ultraviolet (UV) rays makes it possible to increase the maximum resolution of the microscope. The main advantage of the method is that particles of many substances, transparent in visible light, strongly absorb UV radiation of certain wavelengths and are therefore easily distinguishable in UV images. Many substances contained in plant and animal cells (purine bases, pyrimidine bases, most vitamins, aromatic amino acids, some lipids, thyroxine, etc.) have characteristic absorption spectra in the UV region.
Since ultraviolet rays are invisible to the human eye, images in UV microscopy are recorded either photographically or using an electron-optical converter or fluorescent screen. The drug is photographed in three wavelengths of the UV spectrum. Each of the resulting negatives is illuminated with a specific color of visible light (for example, blue, green, and red), and they are all simultaneously projected onto a single screen. The result is a color image of the object in conventional colors, depending on the absorption capacity of the drug in ultraviolet light.
Microphotography and microcinema- this is the acquisition of images on photosensitive layers using a microscope. This method is widely used in conjunction with all other methods of microscopic examination. For microphotography and microcinema, some restructuring of the optical system of the microscope is required - different from visual observation of the focusing of the eyepiece relative to the image given by the lens. Microphotography is necessary when documenting research, when studying objects in UV and IR rays invisible to the eye (see above), as well as objects with low luminescence intensity. Microfilm photography is indispensable in the study of processes that unfold over time (the vital activity of tissue cells and microorganisms, crystal growth, the occurrence of simple chemical reactions, etc.).
Interference contrast method
The interference contrast method (interference microscopy) consists of splitting each beam as it enters the microscope. One of the resulting rays is directed through the observed particle, the other - past it along the same or additional optical branch of the microscope. In the eyepiece part of the microscope, both beams are again connected and interfere with each other. The condenser and lens are equipped with birefringent plates, of which the first splits the original light beam into two beams, and the second reunites them. One of the rays, passing through the object, is delayed in phase (acquires a path difference compared to the second ray). The magnitude of this delay is measured by a compensator. This method makes it possible to observe transparent and colorless objects, but their images can also be multi-colored (interference colors). This method is suitable for studying living tissues and cells and is used in many cases for this purpose. The interference contrast method is often used in conjunction with other microscopy methods, in particular with observation in polarized light. Its use in combination with ultraviolet microscopy makes it possible, for example, to determine the content of nucleic acids in the total dry mass of an object.
Research method in luminescence light
Method studies in the light of luminescence consists of observing under a microscope the green-orange glow of micro-objects, which occurs when they are illuminated with blue-violet light or ultraviolet rays invisible to the eye. Two light filters are introduced into the optical circuit of the microscope. One of them is placed in front of the condenser. It transmits radiation from the illuminator source only at those wavelengths that excite the luminescence of either the object itself (intrinsic luminescence) or special dyes introduced into the preparation and absorbed by its particles (secondary luminescence). The second light filter, which is installed after the lens, transmits only luminescence light to the observer’s eye (or to the photosensitive layer). Fluorescent microscopy uses illumination of preparations both from above (through the lens, which in this case also serves as a condenser) and from below, through a regular condenser. The method has found wide application in microbiology, virology, histology, cytology, in the food industry, in soil research, in microchemical analysis, and in flaw detection. This variety of applications is explained by the very high color sensitivity of the eye and the high contrast of the image of a self-luminous object on a dark, non-luminescent background.
Replica method
The replica method is used to study the surface geometric structure of massive bodies. An imprint is taken from the surface of such a body in the form of a thin film of carbon, collodion, formvar, etc., repeating the surface relief and examined in a transmission electron microscope. Usually, at a sliding (small to the surface) angle, a layer of heavy metal that strongly scatters electrons is sprayed onto the replica in a vacuum, shading the protrusions and depressions of the geometric relief.
Decoration method
The decoration method examines not only the geometric structure of surfaces, but also microfields caused by the presence of dislocations, accumulations of point defects, growth stages of crystalline faces, domain structure, etc. According to this method, a very thin layer of decorating particles (Au atoms) is first deposited onto the sample surface , Pt, etc., molecules of semiconductors or dielectrics), deposited mainly in areas where microfields are concentrated, and then a replica with inclusions of decorating particles is removed.
To obtain cell fractions, various types of centrifugation are widely used: differential centrifugation, zonal centrifugation and equilibrium density gradient centrifugation. Theoretical and practical issues related to centrifugation are comprehensively discussed in Sykes's review.
Differential centrifugation
In the case of differential centrifugation, samples are centrifuged for a certain time at a given speed, after which the supernatant is removed. This method is useful for separating particles that vary widely in sedimentation rates. For example, centrifugation for 5-10 minutes at 3000-5000 d leads to the sedimentation of intact bacterial cells, while the majority of cell fragments remain in the supernatant. Cell wall fragments and large membrane structures can be pelleted by centrifugation at 20,000-50,000 § for 20 minutes, while small membrane vesicles and ribosomes require centrifugation at 200,000 § for 1 hour to pellet.
Zonal centrifugation
Zonal centrifugation is an effective method for separating structures that have similar buoyant densities but differ in particle shape and mass. Examples include the separation of ribosomal subunits, different classes of polysomes, and DNA molecules of different shapes. Centrifugation is carried out either in bucket rotors or in specially designed zonal rotors; To prevent convection during centrifugation, a weak gradient (usually sucrose) is created in the bucket rotor beakers or in the zonal rotor chamber. The sample is applied in the form of a zone or narrow strip at the very top of the gradient column. For subcellular particles, a sucrose gradient of 15 to 40% (w/v) is typically used.
Laue method
used for single crystals. The sample is irradiated by a beam with a continuous spectrum; the mutual orientation of the beam and the crystal does not change. The angular distribution of diffracted radiation has the form of individual diffraction spots (Lauegram).
Debye-Scherrer method
Used to study polycrystals and their mixtures. The random orientation of the crystals in the sample relative to the incident monochromatic beam turns the diffracted beams into a family of coaxial cones with the incident beam on the axis. Their image on photographic film (debyegram) has the form of concentric rings, the location and intensity of which allows us to judge the composition of the substance under study.
Cell culture method
Some tissues can be divided into individual cells so that the cells remain alive and are often able to reproduce. This fact definitively confirms the idea of the cell as a living unit. A sponge, a primitive multicellular organism, can be separated into cells by rubbing it through a sieve. After some time, these cells reconnect and form a sponge. Animal embryonic tissues can be made to dissociate using enzymes or other means that weaken the bonds between cells.
American embryologist R. Garrison (1879-1959) was the first to show that embryonic and even some mature cells can grow and multiply outside the body in a suitable environment. This technique, called cell culturing, was perfected by the French biologist A. Carrel (1873-1959). Plant cells can also be grown in culture, but compared to animal cells they form larger clumps and are more firmly attached to each other, so tissues are formed as the culture grows, rather than individual cells. In cell culture, an entire adult plant, such as a carrot, can be grown from a single cell.
Microfigure method
Using a micromanipulator, individual parts of the cell can be removed, added, or modified in some way. A large amoeba cell can be divided into three main components - the cell membrane, cytoplasm and nucleus, and then these components can be reassembled to form a living cell. In this way, artificial cells consisting of components of different types of amoebas can be obtained.
If we take into account that it seems possible to synthesize some cellular components artificially, then experiments in assembling artificial cells may be the first step towards creating new forms of life in the laboratory. Since every organism develops from a single cell, the method of producing artificial cells in principle allows the construction of organisms of a given type, if at the same time using components slightly different from those found in existing cells. In reality, however, complete synthesis of all cellular components is not required. The structure of most, if not all, components of a cell is determined by nucleic acids. Thus, the problem of creating new organisms comes down to the synthesis of new types of nucleic acids and their replacement of natural nucleic acids in certain cells.
Cell fusion method
Another type of artificial cells can be obtained by fusing cells of the same or different species. To achieve fusion, cells are exposed to viral enzymes; in this case, the outer surfaces of two cells are glued together, and the membrane between them is destroyed, and a cell is formed in which two sets of chromosomes are enclosed in one nucleus. It is possible to fuse cells of different types or at different stages of division. Using this method, it was possible to obtain hybrid cells of a mouse and a chicken, a human and a mouse, and a human and a toad. Such cells are hybrid only initially, and after numerous cell divisions they lose most of the chromosomes of either one or the other type. The final product becomes, for example, essentially a mouse cell with no or only a trace amount of human genes present. Of particular interest is the fusion of normal and malignant cells. In some cases, hybrids become malignant, in others they do not, i.e. both properties can manifest themselves as both dominant and recessive. This result is not unexpected, since malignancy can be caused by various factors and has a complex mechanism.
cell microscopy light
Annex 1
Figure 2. Cryotome Figure 3. Phase contrast microscope
Figure 4. Interference microscope
Posted on Allbest.ru
...Similar documents
Study of the structure and operating principles of light and electron microscopes. Consideration of dark and bright field techniques, phase contrast microscopy, interference and polarization. Vital fixed cell study. Fundamentals of electron microscopy.
lecture, added 05/16/2014
Scanning tunnel microscope, application. Operating principle of an atomic force microscope. Study of biological objects - macromolecules (including DNA molecules), viruses and other biological structures using atomic force microscopy.
course work, added 04/28/2014
The concept of electron microscopy as a set of methods for studying the microstructures of bodies and their local composition using electron microscopes. The content of the television principle of scanning a thin beam of electrons or ions over the surface of a sample.
presentation, added 08/22/2015
Measuring the size of small objects. Phase contrast method. The concept of electron optics. Creation of an electron microscope. Experiments on electron diffraction. Research of the surface geometric structure of cells, viruses and other microobjects.
presentation, added 05/12/2017
Electron microscopic research method. Physical foundations of scanning electron microscopy. Scheme of a scanning electron microscope, the purpose of its components and their functioning. Preparation of objects for research and special requirements for them.
course work, added 05/04/2011
Optical spectrum range. Theoretical foundations of optical NDT methods. Light vibrations. Classification of optical NDT methods. Discrete emission spectrum of gases and liquids. Continuous spectrum of intrinsic radiation of solids with different temperatures.
abstract, added 01/15/2009
General characteristics of the methods used to measure the parameters of capillaries of dies: holographic interferometry, Fourier optics, microscopic. Comparative analysis of the considered methods, determination of their main advantages and disadvantages.
test, added 05/20/2013
Creation of an atomic force microscope, operating principle, advantages and disadvantages. Atomic force microscopy methods. Technical capabilities of the atomic force microscope. Application of atomic force microscopy to describe the deformations of polymer films.
course work, added 11/14/2012
The history of the microscope - a device for obtaining magnified images of objects invisible to the naked eye. Light microscopy methods. The principle of operation and structure of a metallographic microscope. Methods for microscopic examination of metals.
abstract, added 06/10/2009
Fundamentals of scanning electron microscopy. Methodological features of electron microscopic examination of metal melts. Features of microscopes designed to study the structure of surface layers of metal melts.
E. Dark-field microscopy.
18. A microscope consists of optical and mechanical parts. What are optical parts?
A. Tube, eyepiece, condenser
B. Revolver, macro- and microscrew, mirror
C. Revolver, eyepiece
D. Eyepiece, condenser, objective
E. Tube, eyepiece, revolver
19. When using ultraviolet rays as a light source, the resolution of the microscope increases. Which microscopic devices use this light source?
A. Dark-field and luminescent
B. Luminescent, ultraviolet
S. Light and electronic
D.Phase contrast, ultraviolet
E. Polarizing, ultraviolet
20. A microscope consists of mechanical and optical parts. Which parts of a microscope have a diaphragm?
A. Eyepiece and objective
B. Eyepiece and condenser
C. Tube and eyepiece
D. Lens and condenser
E. Tube, lens, eyepiece
21. The experiment used living objects in which it is necessary to determine a number of chemical components using vital observation. What microscopic examination method will be used?
A. Phase contrast microscopy
B. Electron microscopy
C. Fluorescence microscopy
E Dark field microscopy.
22. For histological examination of cells, phosphors were used. What type of microscopy was used in this case?
A. Light microscopy
B. Electron microscopy
C. Fluorescence microscopy
D. Polarization microscopy
E. Dark-field microscopy.
23. The researcher is tasked with obtaining a spatial understanding of the structures of the object being studied. What microscopic instrument will the specialist work with?
A. Ultraviolet microscopy,
B. Phase contrast microscopy,
C. Transmission electron microscopy,
D. Scanning electron microscopy,
E. Polarization microscopy
24. Mercury-quartz lamps are used as a light source. What is the resolution of the microscope with this light source?
25. The resolution of a microscope depends on the wavelength of the light source. What is the resolving power of a light microscope?
26. Before starting the examination of a histological specimen, it is necessary to uniformly illuminate the field of view. What parts of the microscope are used for this?
A. Micro- and macrovit
B. Condenser and mirror
C. Tube and tube holder
D. Tube and eyepiece
27. The researcher was tasked with studying the ultra-microscopic structure of the erythrocyte plasmalemma. What microscopic instrument will be used?
A. Svetova
B. Phase contrast
C. Electronic
D. Polarization
E. Ultraviolet
28. When studying skeletal muscle tissue, it is necessary to determine the iso- and anisotropic structures of the tissue. What type of microscopy will be used?
A. Svetova
B. Phase contrast
C. Electronic
D. Polarization
E. Ultramicroscopic
29. The resolution of a fluorescent microscope depends on the wavelength of the light source. What is it equal to?
A. 0.1 µm C. 0.4 µm
B. 0.2 µm D. 0.1 nm
30. In a clinical laboratory, microscopic examinations are used to study a complete blood count. What microscope is needed for this?
A. Svetovoy,
B. Phase contrast,
S. Electronic,
D. Polarization,
E. Ultraviolet.
31. A living object with natural luminescence is presented for research. What type of microscopy should be used for this study?
A. Svetova
B. Phase contrast
C. Electronic
D. Polarization
E. Ultraviolet
32. As a result of a biopsy, tumor cell material was obtained. It is necessary to study their ultramicroscopic structure. What type of microscopy is used in this study?
A. Svetova
B. Phase contrast
C. Electronic
D. Polarization
E. Ultraviolet
TOPIC 2: HISTOLOGICAL TECHNIQUE
Basic principles of preparing preparations for light and electron microscopy, taking material (biopsy, needle puncture biopsy, autopsy). Fixation, dehydration, compaction of objects, preparation of sections on microtomes and ultramicrotomes. Types of micropreparations - section, smear, imprint, film, thin section. Staining and contrasting preparations. The concept of histological dyes.
Microscopic technique.
The main stages of cytological and histological analysis:
Selecting a research object
Preparing it for examination under a microscope
Application of microscopy methods
Qualitative and quantitative analysis of acquired images
Methods used in histological technology:
1. Lifetime.
2. Posthumous.
I INTERVITUAL METHODS
The purpose of intravital research is to obtain information about the life of a cell: movement, division, growth, differentiation, cell interaction, life expectancy, destruction, reactive changes under the influence of various factors.
The study of living cells and tissues is possible outside the body (in vitro) or inside the body (in vivo).
A. Study of living cells and tissues in culture (in vitro) –
Cultivation method
There are: a) suspension cultures (cells suspended in a nutrient medium), b) tissue, c) organ, d) monolayer.
The method of culturing tissue outside the body is the most common. Tissue can be cultivated in special transparent hermetically sealed chambers. Under sterile conditions, a drop of nutrient medium is placed into the chamber. The best nutrient medium is blood plasma, to which embryonic extract is added (an extract from the tissues of the embryo containing a large amount of substances that stimulate growth). A piece of organ or tissue (no more than 1 mm3) that needs to be cultured is also placed there.
The cultured tissue should be maintained at the body temperature of the organism whose tissue was taken for research. Since the nutrient medium quickly becomes unusable (decomposition products released by the cultured tissue accumulate in it), it needs to be changed every 3-5 days.
The use of the cultivation method made it possible to identify a number of patterns of differentiation, malignant degeneration of cells, interactions of cells with each other, as well as with viruses and microbes. Cultivation of embryonic tissues made it possible to study the development of bone, cartilage, skin, etc.
The cultivation method is of particular importance for conducting experimental observations on human cells and tissues, in particular for determining sex, malignant degeneration, hereditary diseases, etc.
Disadvantages of the method:
1. The main disadvantage of this method is that the tissue or organ is examined in isolation from the body. Without experiencing the neurohumoral influence of the body, it loses its inherent differentiation.
2. The need for frequent transplants (with long-term cultivation).
3. Identical refractive index of tissues.
Related information.
Polarization microscopy- one of the highly effective methods of morphological research, which has broad capabilities for identifying biological structures, which, combined with accessibility and relative simplicity, determines its high value. The method makes it possible to study not only the histological structure of the drug, but also some of its histochemical parameters. In the 40s and 50s of the XX century. polarization microscopy was considered an ultrastructural method, since it made it possible to see the ultrastructural abilities of tissues.
Polarization microscopy is designed to study the properties of histological structures that have the ability of birefringence (anisotropy) - the splitting of a light beam when passing through an anisotropic medium. A light wave in an anisotropic medium breaks up into two waves with mutually perpendicular planes of oscillation of electromagnetic waves. These planes are called planes of polarization. Polarized light differs from ordinary (non-polarized) light in that in the latter the light waves oscillate in different planes, while in polarized light they occur only in a certain plane.
To create the polarization effect, a polarizing microscope uses two polaroids. The first, which is called a polarizer, is placed between the microscope illuminator and the histological specimen. The second polaroid, located between the histological specimen and the researcher’s eye, is the analyzer. Both the polarizer and the analyzer are optically exactly the same polarizing filters, so they can be swapped (if the design of the microscope allows this). Previously, Nicolas, Arens or Thomson prisms made from Iceland spar were used for polarization microscopy. These prisms had a limited angle of refraction of light. Currently, instead of them, flat polarizing filters are used, producing wide-field polarized light.
In creating polarized light, the determining role is played by the relative position of the polarizer and analyzer relative to the optical axis of the microscope. If they are oriented in such a way that both transmit polarized light in the same plane, i.e. when their planes of polarization coincide, both polarizing filters are capable of transmitting polarized light; the field of view of the microscope is bright (Fig. 1a).
Rice. 1 Brightfield specimen of a human lung, OlympusCX41, 10x lens
If the polarization planes of the polarizing filters are mutually perpendicular (this is achieved by rotating the analyzer 90° around the optical axis of the microscope), then the polarized light does not pass through and the researcher sees a dark field of view (Fig. 2).
When the polarizer is rotated 360° as it rotates, the field of view is completely darkened twice and completely brightened twice. In the past, compensatory Bernauer filters have been used, which produce a reddish tint to the dark field of view ( U-TP530 ). When black mirror filters are used, the darkened field of view does not appear completely dark, but rather faintly illuminated.
Fig. 2 Human lung specimen in polarized light, 10x objective
In cases where, with a crossed position of polarizing filters (i.e. in orthoscopy), anisotropic substances contained in a histological specimen are encountered in the path of polarized light, these substances split the polarized light into two beams with mutually perpendicular planes of oscillation of light waves. Light rays with a plane of vibration coinciding with the plane of polarization pass through the analyzer, and those with a plane perpendicular are cut off, as a result of which the intensity of the light flux entering the researcher’s eye and onto the camera is only half the intensity of the original light beam. As a result of the described processes, anisotropic substances located between two crossed polarizers are visible against a dark background in the form of light luminous objects. At the same time, isotropic structures that do not have the ability of birefringence remain dark.
This also influences the choice cameras for polarization microscopy. Since the task is to capture small bright signals on a dark background, usually a camera for bright-field microscopy may not be enough due to the low sensitivity of the camera and the large amount of noise that is generated during recording. For polarizing microscopy A microscopy camera with high sensitivity and accurate color reproduction is required. It is preferable to use cameras based on CCD matrices (, VZ-CC50S), however, at the current stage, you can also use budget versions of cameras based on Sony IMX series CMOS matrices ().
Biological tissues contain a sufficient number of anisotropic structures: elements of the contractile apparatus of muscles, amyloid, uric acid, collagen formations, some lipids, a number of crystals, etc.
Light rays split in an anisotropic object and passing through the analyzer are characterized by unequal wave propagation speeds. Depending on the magnitude of this difference (it is also called delay value of the light beam) and due to differences in light absorption in the analyzer, the glow of anisotropic objects can be white or colored. In the latter case we are talking about the phenomenon of dichroism ( double absorption I). When studied in a polarized field, color effects are produced, for example, by many crystals.
The process of birefringence can be enhanced by the use of certain dyes, the molecules of which have the ability to be orientedly deposited on anisotropic structures. Histochemical reactions that result in the anisotropy effect are called topo-optic reactions (G. Romhanyi). There are two types of such reactions - additive and inverse. With additive reactions, the delay of the light beam increases, which is called positive anisotropy; with inverse reactions, it decreases - negative anisotropy.
HARDWARE AND EQUIPMENT
Polarization microscopy is carried out using special polarizing microscopes. As an example, we can name imported microscopes. Most modern optical microscopes are equipped with accessories for polarization microscopy.
Any laboratory or research grade light microscope can be used for polarization microscopy. It is enough to have two polarizing filters, one of which, acting as a polarizer, is placed between the light source and the specimen, and the other, which plays the role of an analyzer, is placed between the specimen and the researcher’s eye. The polarizer can be built into the condenser or placed underneath it above the field diaphragm, and the analyzer can be placed in a slot in the revolver or in an intermediate insert.
In Fig. Figure 3 shows a schematic diagram of a polarizing microscope. In addition to the components common to all light microscopes, a polarizing microscope has two polarizing filters (a polarizer, usually located under the condenser, and an analyzer located in the eyepiece), as well as a compensator. The analyzer must rotate, and an appropriate graduated scale is required to determine the degree of rotation.
A polarizing microscope uses an illumination source that provides a high density of light beam. It is recommended to use a 100 W lamp at a voltage of 12 V as such a source. For some types of research, monochromatic light is required. For this purpose, a metal interference filter is used, which is best placed above the mirror. Light-scattering frosted glass is placed in front of the polarizer, i.e. between it and the light source, but in no case after the polarizer, since this will disrupt the function of the polarizing filter.
In the past, achromatic objectives without internal tension were used for polarization microscopy, but these are now rare. Today, only plan achromatic objectives, which do not have internal tensions, are used in polarizing microscopes. Apochromatic lenses can be used only in cases where normal color rendition is required during microphotography.
Polarizing microscopes are equipped with a rotating stage, the position of which relative to the optical axis can be changed. The angle of rotation of the table is measured using a degree scale marked along its circumference. One of the prerequisites for the effective use of polarization microscopy is careful alignment of the rotating stage using centering screws.
An important element of a polarizing microscope is a compensator placed between the objective and the analyzer, usually in the microscope tube. The compensator is a plate made from special types of gypsum, quartz or mica. It allows you to measure the difference in the path of split light rays, expressed in nanometers. The functioning of the compensator is ensured by its ability to change the difference in the path of light rays, reducing it to zero or increasing it to the maximum. This is achieved by rotating the compensator around the optical axis.
MICROSCOPY TECHNIQUE IN POLARIZED LIGHT
It is more convenient to carry out polarization microscopy in a darkened room, since the intensity of the light flux entering the researcher’s eye is reduced by 2 times compared to the original one. After turning on the microscope illuminator, first achieve the brightest possible illumination of the field of view by rotating the polarizer or analyzer. This position of the polarization filters corresponds to the coincidence of their planes of polarization. The drug is placed on a stage and studied first in a bright field. Then, by rotating the polarizer (or analyzer), the field of view is darkened as much as possible; this filter position corresponds to the perpendicular arrangement of the planes of polarization. In order to reveal the effect of anisotropy, it is necessary to combine the plane of polarization of an anisotropic object with the plane of polarized light. Empirically, this is achieved by rotating the object stage around the optical axis. If a light microscope that is not equipped with a rotating stage is used for polarization microscopy, then the histological specimen must be rotated manually. This is acceptable, but in this case it is impossible to carry out certain types of polarization microscopy that require quantitative assessment (determining the sign of birefringence, the magnitude of the difference in the path of light rays).
If anisotropic objects in the test specimen are arranged in an orderly manner (for example, anisotropic disks of striated muscle fibers), it is convenient to study them in a fixed position of the stage, in which these objects give maximum luminescence against a dark background. If anisotropic structures are located chaotically in the preparation (for example, crystals), then when studying them you have to constantly rotate the stage to achieve the glow of one or another group of objects.
To carry out a more in-depth analysis and evaluation of topo-optical reactions, it is necessary to know the methodology for determining the relative sign of birefringence, the magnitude of the difference in the path of the rays and the index (coefficient) of refraction.
The sign of birefringence characterizes the degree and direction of displacement of the path of light rays passing through the analyzer. This shift is caused by topo-optical dyes, and if it is directed towards reducing the difference in the path of the rays, they speak of a negative sign of birefringence ( negative anisotropy), if it helps to increase the difference in the path of the rays, then a positive sign of birefringence is stated ( positive anisotropy). If the difference in the path of the rays disappears, then the anisotropy effect is leveled out.
The sign of birefringence is determined using a compensator. The procedure for its use is as follows. The object under study is placed in a position at which maximum luminescence of anisotropic structures is achieved in a dark field of view. The RI compensator plate is rotated around the optical axis at an angle of +45° relative to the polarization plane of the analyzer. An object, depending on the difference in the path of light rays, which can range from 20 to 200 nm, acquires either a blue or yellow color. In the first case, the sign of birefringence is positive, in the second - negative. It should be borne in mind that in the case when the compensator is located at an angle of +45°, the overall background of the darkened field of view has a red tint.
A λ/4 compensator (U-TP137) can also be used. The procedure for using it is the same, only the field of view has a gray tint rather than red, and the object glows with a positive sign of refraction, and is darkened with a negative sign.
Quantitative determination of the difference in the path of light rays, expressed in nanometers, is carried out using a Braque Köhler compensator. To do this, use the formula:
Γ=Γλ×sinφ
where λ is a constant marked on the compensator by the manufacturer, φ is the angle of rotation of the compensator relative to the polarization plane of the analyzer.
The refractive index of an anisotropic object is determined by comparing it (under a microscope) with a test object placed next to it. Standard liquids with a known refractive index are used as test objects. The object and sample are placed side by side on the stage. When their refractive indices do not match, a light line called the Beck line is visible between the object and the sample. Raising the microscope tube relative to the focused position causes a shift of the Beck line towards the medium, which gives a more pronounced effect of refraction. When the refractive indices of the object and the sample coincide, the Beck line disappears. Typically, the refractive index is determined in monochromatic light for the sodium line of the spectrum (at a wavelength of 589 nm and a temperature of 20 ° C). Refraction should be determined for two mutually perpendicular planes of polarization. For this purpose, the analyzer is removed and the refraction of the object is recorded in its two mutually perpendicular positions. The difference between both refractive indices (ng - nk) characterizes the strength of refraction.
FEATURES OF MATERIAL PROCESSING AND PREPARATION OF PREPARATIONS
Fixing material for polarization microscopy in acidic formalin is undesirable, since the formalin pigment formed by the interaction of tissue hemoglobin with acidic formaldehyde has anisotropic properties and makes it difficult to study preparations in polarized light. G. Scheuner and J. Hutschenreiter (1972) recommend using 10% neutral formalin, Baker's calcium-formol solution, and Carnoy's liquid for this purpose.
The duration of fixation in 10% neutral formalin is 24 - 72 hours at 4 °C, in Baker's calcium-formol solution - 16 - 24 hours at 4 °C. Fixation in calcium-formol is especially preferred when studying lipid-protein compounds. Carnoy's liquid quickly saturates fabrics. Pieces with a thickness of 1 - 2 mm can be profiled after just 1 hour at a temperature of 4 °C. Fixation in Carnoy's fluid is not suitable for lipid studies. In addition, Zenker's liquid is used, especially when impregnated with gold and silver salts. After treatment with a mixture of Zenker's liquid and acetic acid, red blood cells acquire the ability to undergo birefringence.
When examining dense tissues (bones, teeth) in a polarizing microscope, in addition to acid decalcification, additional processing is required to remove collagen fibers. For this purpose, sections of such tissues are boiled for several minutes in a mixture of glycerin and potassium hydroxide (10 ml of glycerin and 2 grains of potassium hydroxide) until completely whitened, then the alkali is carefully drained, the section is washed in water and transferred with tweezers to the microscope stage.
For polarization microscopy, paraffin, frozen and cryostat sections are used. Unstained frozen sections for examination under polarized light are embedded in glycerol. Unfixed cryostat sections are suitable for polarization microscopic analysis immediately after preparation. Due to their high sensitivity to the damaging effects of various environmental factors, these sections are still recommended to be fixed in 10% neutral formaldehyde or calcium-formol solution.
The results of polarization microscopy are influenced by the thickness of histological sections. When studying thick sections, conditions are created for the superposition of different anisotropic structures on top of each other. In addition, with different slice thicknesses, the anisotropic properties of the structures being studied may change, so it is very important, especially in comparative studies, to ensure a constant slice thickness. The recommended maximum section thickness should not exceed 10 µm.
Another mandatory condition is careful dewaxing of the sections, since unremoved paraffin residues give a pronounced anisotropy effect, complicating the study. Paraffin lingers especially long on red blood cells and cell nuclei. In order to completely remove paraffin from the sections, it is recommended to carry out the following processing.
- Xylene 30 min
- Alcohol 100% 5 min
- A mixture of methanol and chloroform (1:1) at 50 °C for 24 hours
- Alcohol 100% 5 min
- Alcohol 70% 10 min Water
It should also be kept in mind that sections that are subjected to polarization microscopy should not come into contact with phenols (for example, they should not be cleared in carbolic xylene).
More detailed information on polarization microscopy and the use of compensators can be obtained from the link (http://www.olympusmicro.com/primer/techniques/polarized/polarizedhome.html).
If you have any questions about polarization microscopy, please contact the School of Microscopy.
Of all the variety of devices for microscopy, polarizing microscopes are the most technically complex. Such attention to the design of the device in terms of manufacturability is due to the need to obtain images of the highest quality, which are directly influenced by the design of the optical and lighting parts of the microscope. The main area of use of polarizing devices for microscopy is the study of minerals, crystals, slags, anisotropic objects, textiles and refractory products, as well as other materials that are characterized by birefringence. The latter principle is used to form images in microscopy devices in which the sample under study is irradiated with polarizing rays. In this case, the anisotropic properties of the samples appear after changing the direction of the beam. For these purposes, the design of polarizing microscopes includes field filters rotating in different planes relative to each other: the analyzer rotates 180 degrees, and the polarizer rotates 360. The main feature of devices for microscopy in polarized light is the ability to conduct orthoscopic and conoscopic studies, which are not available with most other types of microscopes.
Studying a sample under a polarizing microscope begins with installing a polarizer in the illumination part of the microscope under the condenser, next to the aperture diaphragm. In this case, the analyzer is located between the eyepiece and the lens - behind the latter along the path of the light rays. With the correct setup of such a device for microscopy, after crossing the filter fields, the visible field will be uniformly dark, forming the so-called extinction effect. Upon completion of the device settings, the sample under study is fixed on the stage and studied. The tables of polarizing microscopes are centered relative to the optical axis and can be rotated 360 degrees, and in similar devices for laboratory and research purposes they also have a vernier. The optics and lighting system of polarizing microscopes are of the highest quality and such manufacturing precision that allows you to obtain the clearest possible image without distortion. Often, the set of devices for studying samples in polarized light includes a compensator and a Bertrand lens. The first makes it possible to effectively study the structure of minerals, and the lens allows you to enlarge and focus the observation area when image changes occur after rotating the stage. Today there are three main types of such devices for microscopy on the market - the already mentioned research and laboratory microscopes, as well as a working polarizing microscope.