MICROSCOPE

MICROSCOPE. MICROSCOPIC EQUIPMENT. HISTOLOGIC TECHNIQUE. CYTOLOGY. GENERAL STRUCTURE OF THE CELL. SUPERFICIAL COMPLEX

 

Using lectures (on the web-page of the department posted the presentation text and lectures), books, additional literature and other sources, students must to prepare the following theoretical questions:

1. Rules of working with light microscope.

2. Rules of histologic specimen investigation under light microscope.

3. Basic steps of specimen’s preparation.

4. Histologic dyes classification.

5. Methods of histologic investigation: a light microscopy, an electron microscopy, histochemistry, an immune histochemistry, morphometry, radioavtography.

6. Living substance organization forms.

7. Shwann cell theory.

8. The term of the cell, its structural components.

9. Cell membrane ultrastructure and functions.

10. Types of intercellular junctions (adhesive, isolating, and communicative).

11. Noncellular structures (symplast, syncytium and intercellular substance) morphofunctional features.

 

The objective of a histology course is to lead the student to understand the microanatomy of cells, tissues, and organs and to correlate structure with function.

Modern histology is characterized by a wide usage of complex modern methods of investigation: microscopic, histochemical, electron microscopy, skanning, radioavtography, living cells method, tissues and organs transplantation. They give the possibility to study in detail the structure and functions of the cells and organs under normal conditions and in pathology. Each doctor has to know all the stages of modern methods of histologic investigation

Histological examination of tissues starts with surgery, biopsy or autopsy (or necropsy, in the case of animal tissues). Engineered Tissue is also used for histological examination, some labs are able to use cells and engineer a tissue that would act as an equivalent to real tissue. The equivalent would not resemble real tissue, but would be a useful model for studying the interactions of cells with infection and many other conditions.

 

The methods used by histologists are extremely diverse. Much of the histology course content can be framed in terms of light microscopy. Today, students in histology laboratories use either light microscopes or, with increasing frequency, virtual microscopy, which represents a method of viewing a digitized microscopic specimen on a computer screen. In the past, more detailed interpretation of microanatomy was with the electron microscope (EM) — both the transmission electron microscope (TEM) and the scanning electron microscope (SEM). Now the atomic force microscope (AFM) can also provide high-resolution images, which are comparable in resolution to those obtained from TEM. Both EM and AFM, because of their greater resolution and useful magnification, are often the last step in data acquisition from many auxiliary techniques of cell and molecular biology.

These auxiliary techniques include:

• histochemistry and cytochemistry,

• immunocytochemistry and hybridization techniques,

• autoradiography,

• organ and tissue culture,

• cell and organelle separation by differential centrifugation,

and

• specialized microscopic techniques and microscopes.

 

Main steps of preparing of tissue specimens for

a light microscopy

1.     Selection and fixation of the material for investigation (formalin, ethyl alcohol, special mixtures).The tissues are mechanically and biochemically stabilized in a fixative.

Processing includes the next steps.

2.     The most common technique is wax processing. The samples are immersed in multiple baths of progressively more concentrated ethanol to dehydrate the tissue, followed by a clearing agent such as, xylene or Histoclear, and finally hot molten paraffin wax (impregnation). During this 12 to 16 hour process, paraffin wax will replace the xylene:

3.     Dehydration (substitutions) in ethyl alcohol.

4.     Clearing (xylene).

5.     Infiltration (ethyl alcohol, xylol).

6.     Embedding (in paraffin or celloidin).       Soft, moist tissues are turned into a hard paraffin block, which is then placed in a mold containing more molten wax (embedded) and allowed to cool and harden. Embedding can also be accomplished using frozen, non-fixed tissue in a freezing medium. This freezing medium is liquid at room temperature but when cooled will solidify. Non-fixed tissue allows for procedures such as in-situ hybridizations for specific mRNA that would have been destroyed during the fixing process. It also allows for very short turnaround where that is needed, as with a patient currently undergoing surgery.

7.     Sectioning with microtome (6-8 mkm thick).     The tissue is then sectioned into very thin (2 – 8 micrometer) sections using a microtome. These slices, usually thinner than the average cell, are then placed on a glass slide for staining.

         Frozen tissue embedded in a freezing medium is cut on a microtome in a cooled machine called a cryostat.

8.     Mounting (glass slide).

9.     Removal of paraffin.

10. Rehydration.

11. Staining and covering.Routine staining:This is done to give contrast to the tissue being examined, as without staining it is very difficult to see differences in cell morphology. Hematoxylin and eosin (abbreviated H&E) are the most commonly used stains in histology and histopathology. Hematoxylin colors nuclei blue, eosin colors the cytoplasm pink. To see the tissue under a microscope, the sections are stained with one or more pigments. Special Staining: There are hundreds of various other techniques which have been used to selectively stain cells and cellular components. Other compounds used to color tissue sections include safranin, oil red o, congo red, fast green FCF, silver salts and numerous natural and artificial dyes, that were usually originated from the development dyes for the textile industry.

 

Hematoxylin and Eosin Staining With Formalin Fixation

The routinely prepared hematoxylin and eosin–stained

section is the specimen most commonly studied. The slide set given each student to study with the light microscope consists mostly of formalin-fixed, paraffin-embedded, hematoxylin and eosin (H&E)–stained specimens.

The first step in preparation of a tissue or organ sample is fixation to preserve structure. Fixation, usually by a chemical or mixture of chemicals, permanently preserves the tissue structure for subsequent treatments. Specimens should be immersed in fixative immediately after they are removed from the body.

Fixation is used to:

• terminate cell metabolism,

• prevent enzymatic degradation of cells and tissues by autolysis (self-digestion),

• kill pathogenic microorganisms such as bacteria, fungi, and viruses, and

• harden the tissue as a result of either cross-linking or denaturing protein molecules.

Formalin, a 37% aqueous solution of formaldehyde, at various dilutions and in combination with other chemicals and buffers, is the most commonly used fixative. Formaldehyde preserves the general structure of the cell and extracellular components by reacting with the amino groups of proteins (most often cross-linked lysine residues). Because formaldehyde does not significantly alter their three-dimensional structure, proteins maintain their ability to react with specific antibodies. This property is important in immunocytochemical staining methods. The standard commercial solution of formaldehyde buffered with phosphates (pH 7) acts relatively slowly but penetrates the tissue well. However, because it does not react with lipids, it is a poor fixative of cell membranes.

In the second step, the specimen is prepared for embedding in paraffin to permit sectioning.

Preparing a specimen for examination requires its infiltration with an embedding medium that allows it to be thinly sliced, typically in the range of 5 to 15 μm (1 micrometer [μm] equals 1/1,000 of a millimeter [mm]). The specimen is washed after fixation and dehydrated in a series of alcohol solutions of ascending concentration as high as 100% alcohol to remove water. In the next step, clearing, organic solvents such as xylol or toluol, which are miscible in both alcohol and paraffin, are used to remove the alcohol before infiltration of the specimen with melted paraffin. When the melted paraffin is cool and hardened, it is trimmed into an appropriately sized block. The block is then mounted in a specially designed slicing machine — a microtome — and cut with a steel knife. The resulting sections are then mounted on glass slides using mounting medium (pinene or acrylic resins) as an adhesive.

In the third step, the specimen is stained to permit examination.

Because paraffin sections are colorless, the specimen is not yet suitable for light microscopic examination. To color or stain the tissue sections, the paraffin must be dissolved out, again with xylol or toluol, and the slide must then be rehydrated through a series of solutions of descending alcohol concentration. The tissue on the slides is then stained with hematoxylin in water. Because the counterstain, eosin, is more soluble in alcohol than in water, the specimen is again dehydrated through a series of alcohol solutions of ascending concentration and stained with eosin in alcohol.

After staining, the specimen is then passed through xylol or toluol to a nonaqueous mounting medium and covered with a coverslip to obtain a permanent preparation.

Other Fixatives

Formalin does not preserve all cell and tissue components. Although H&E–stained sections of formalin-fixed specimens are convenient to use because they adequately display general structural features, they cannot elucidate the specific chemical composition of cell components. Also, many components are lost in the preparation of the specimen. To retain these components and structures, other fixation methods must be used. These methods are generally based on a clear understanding of the chemistry involved. For instance, the use of alcohols and organic solvents in routine preparations removes neutral lipids. To retaieutral lipids, such as those in adipose cells, frozen sections of formalin-fixed tissue and dyes that dissolve in fats must be used; to retain membrane structures, special fixatives containing heavy metals that bind to the phospholipids, such as permanganate and osmium, are used. The routine use of osmium tetroxide as a fixative for electron microscopy is the primary reason for the excellent preservation of membranes in electron micrographs.

Other Staining Procedures

Hematoxylin and eosin are used in histology primarily to display structural features.

Despite the merits of H&E staining, the procedure does not adequately reveal certain structural components of histologic sections such as elastic material, reticular fibers, basement membranes, and lipids. When it is desirable to display these components, other staining procedures, most of them selective, can be used. These procedures include the use of orcein and resorcin-fuchsin for elastic material and silver impregnation for reticular fibers and basement membrane material. Although the chemical bases of many staining methods are not always understood, they work. Knowing the components that a procedure reveals is more important than knowing precisely how the procedure works.

 

HISTOCHEMISTRY AND CYTOCHEMISTRY

Specific chemical procedures can provide information about the function of cells and the extracellular components of tissues.

Histochemical and cytochemical procedures may be based on specific binding of a dye, use of a fluorescent dye–labeled antibody with a particular cell component, or the inherent enzymatic activity of a cell component. In addition, many large molecules found in cells can be localized by the process of autoradiography, in which radioactively tagged precursors of the molecule are incorporated by cells and tissues before fixation. Many of these procedures can be used with both light microscopic and electron microscopic preparations. Before discussing the chemistry of routine staining and histochemical and cytochemical methods, it is useful to examine briefly the nature of a routinely fixed and embedded section of a specimen.

 

Chemical Composition of Histologic Samples

The chemical composition of a tissue ready for routine staining differs from living tissue. The components that remain after fixation consist mostly of large molecules that do not readily dissolve, especially after treatment with the fixative. These large molecules, particularly those that react with other large molecules to form macromolecular complexes, are usually preserved in a tissue section. Examples of such large macromolecular complexes include:

• nucleoproteins formed from nucleic acids bound to protein,

• intracellular cytoskeletal proteins complexed with associated proteins,

• extracellular proteins in large insoluble aggregates, bound to similar molecules by cross-linking of neighboring molecules, as in collagen fiber formation, and

• membrane phospholipid–protein (or carbohydrate) complexes.

These molecules constitute the structure of cells and tissues — that is, they make up the formed elements of the tissue. They are the basis for the organization that is seen in tissue with the microscope. In many cases, a structural element is also a functional unit. For example, in the case of proteins that make up the contractile filaments of muscle cells, the filaments are the visible structural components and the actual participants in the contractile process. The RNA of the cytoplasm is visualized as part of a structural component (e.g., ergastoplasm of secretory cells, Nissl bodies of nerve cells) and is also the actual participant in the synthesis of protein.

Many tissue components are lost during the routine preparation of H&E–stained sections.

Despite the fact that nucleic acids, proteins, and phospholipids are mostly retained in tissue sections, many are also lost. Small proteins and small nucleic acids, such as transfer RNA, are generally lost during the preparation of the tissue. As previously described, neutral lipids are usually dissolved by the organic solvents used in tissue preparation. Other large molecules also may be lost, for example, by being hydrolyzed because of the unfavorable pH of the fixative solutions. Examples of large molecules lost during routine fixation in aqueous fixatives are:

• glycogen (an intracellular storage carbohydrate common in liver and muscle cells), and

• proteoglycans and glycosaminoglycans (extracellular complex carbohydrates found in connective tissue).

These molecules can be preserved, however, by using a nonaqueous fixative for glycogen or by adding specific binding agents to the fixative solution that preserve extracellular carbohydrate-containing molecules.

Soluble components, ions, and small molecules are also lost during the preparation of paraffin sections. Intermediary metabolites, glucose, sodium, chloride, and similar substances are lost during preparation of routine H&E paraffin sections. Many of these substances can be studied in special preparations, sometimes with considerable loss of structural integrity. These small soluble ions and molecules do not make up the formed elements of a tissue; they participate in synthetic processes or cellular reactions. When they can be preserved and demonstrated by specific methods, they provide invaluable information about cell metabolism, active transport, and other vital cellular processes. Water, a highly versatile molecule, participates in these reactions and processes and contributes to the stabilization of macromolecular structure through hydrogen bonding.

 

Chemical Basis of Staining Acidic and Basic Dyes

Hematoxylin and eosin are the most commonly used dyes in histology.

An acidic dye, such as eosin, carries a net negative charge on its colored portion and is described by the general formula [Na⁺dye^–].

A basic dye carries a net positive charge on its colored portion and is described by the general formula [dye⁺Cl^–].

Hematoxylin does not meet the definition of a strict basic dye but has properties that closely resemble those of a basic dye.

Basic dyes react with anionic components of cells and tissue (components that carry a net negative charge).

Anionic components include the phosphate groups of nucleic acids, the sulfate groups of glycosaminoglycans, and the carboxyl groups of proteins. The ability of such anionic groups to react with a basic dye is called basophilia. Tissue components that stain with hematoxylin also exhibit basophilia. The reaction of the anionic groups varies with pH.

Thus:

• At a high pH (about 10), all three groups are ionized and available for reaction by electrostatic linkages with the basic dye.

• At a slightly acidic to neutral pH (5 to 7), sulfate and phosphate groups are ionized and available for reaction with the basic dye by electrostatic linkages.

• At low pH (below 4), only sulfate groups remain ionized and react with basic dyes.

Therefore, staining with basic dyes at a specific pH can be used to focus on specific anionic groups; because the specific anionic groups are found predominantly on certain macromolecules, the staining serves as an indicator of these macromolecules.

As mentioned, hematoxylin is not, strictly speaking, a basic dye. It is used with a mordant (i.e., an intermediate link between the tissue component and the dye). The mordant causes the stain to resemble a basic dye. The linkage in the tissue–mordant–hematoxylin complex is not a simple electrostatic linkage; when sections are placed in water, hematoxylin does not dissociate from the tissue. Hematoxylin lends itself to those staining sequences in which it is followed by aqueous solutions of acidic dyes. True basic dyes, as distinguished from hematoxylin, are not generally used in sequences in which the basic dye is followed by an acidic dye. The basic dye then tends to dissociate from the tissue during the aqueous solution washes between the two dye solutions.

Acidic dyes react with cationic groups in cells and tissues, particularly with the ionized amino groups of proteins.

The reaction of cationic groups with an acidic dye is called acidophilia. Reactions of cell and tissue components with acidic dyes are neither as specific nor as precise as reactions with basic dyes. Although electrostatic linkage is the major factor in the primary binding of an acidic dye to the tissue, it is not the only one; because of this, acidic dyes are sometimes used in combinations to color different tissue constituents selectively. For example, three acidic dyes are used in the Mallory staining technique: aniline blue, acid fuchsin, and orange G. These dyes selectively stain collagen, ordinary cytoplasm, and red blood cells, respectively. Acid fuchsin also stains nuclei.

In other multiple acidic dye techniques, hematoxylin is used to staiuclei first, and then acidic dyes are used to stain cytoplasm and extracellular fibers selectively. The selective staining of tissue components by acidic dyes is attributable to relative factors such as the size and degree of aggregation of the dye molecules and the permeability and “compactness” of the tissue.

Basic dyes can also be used in combination or sequentially (e.g., methyl green and pyronin to study protein synthesis and secretion), but these combinations are not as widely used as acidic dye combinations.

A limited number of substances within cells and the extracellular matrix display basophilia.

These substances include:

• heterochromatin and nucleoli of the nucleus (chiefly because of ionized phosphate groups iucleic acids of both),

• cytoplasmic components such as the ergastoplasm (also because of ionized phosphate groups in ribosomal RNA), and

• extracellular materials such as the complex carbohydrates of the matrix of cartilage (because of ionized sulfate groups).

Staining with acidic dyes is less specific, but more substances within cells and the extracellular matrix exhibit acidophilia.

These substances include:

• most cytoplasmic filaments, especially those of muscle cells,

• most intracellular membranous components and much of the otherwise unspecialized cytoplasm, and

• most extracellular fibers (primarily because of ionized amino groups).

Metachromasia

Certain basic dyes react with tissue components that shift their normal color from blue to red or purple; this absorbance change is called metachromasia.

The underlying mechanism for metachromasia is the presence of polyanions within the tissue. When these tissues are stained with a concentrated basic dye solution, such as toluidine blue, the dye molecules are close enough to form dimeric and polymeric aggregates. The absorption properties of these aggregations differ from those of the individual nonaggregated dye molecules. Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups — such as the ground substance of cartilage, heparin-containing granules of mast cells, and rough endoplasmic reticulum of plasma cells—exhibit metachromasia. Therefore, toluidine blue will appear purple to red when it stains these components.

Aldehyde Groups and the Schiff Reagent

The ability of bleached basic fuchsin (Schiff reagent) to react with aldehyde groups results in a distinctive red color and is the basis of the periodic acid–Schiff and Feulgen reactions.

The periodic acid–Schiff (PAS) reaction stains carbohydrates and carbohydrate-rich macromolecules. It is used to demonstrate glycogen in cells, mucus in various cells and tissues, the basement membrane that underlies epithelia, and reticular fibers in connective tissue. The Feulgen reaction, which relies on a mild hydrochloric acid hydrolysis, is used to stain DNA.

The PAS reaction is based on the following facts:

• Hexose rings of carbohydrates contain adjacent carbons, each of which bears a hydroxyl (–OH) group.

• Hexosamines of glycosaminoglycans contain adjacent carbons, one of which bears an –OH group, whereas the other bears an amino (–NH₂) group.

• Periodic acid cleaves the bond between these adjacent carbon atoms and forms aldehyde groups.

• These aldehyde groups react with the Schiff reagent to give a distinctive magenta color.

The PAS staining of basement membrane and reticular fibers is based on the content or association of proteoglycans (complex carbohydrates associated with a protein core). PAS staining is an alternative to silver-impregnation methods, which are also based on reaction with the sugar molecules in the proteoglycans.

Photomicrograph of kidney tissue stained by the PAS method. This histochemical method demonstrates and localizes carbohydrates and carbohydrate-rich macromolecules. The basement membranes are PAS positive as evidenced by the magenta staining of these sites. The kidney tubules (T ) are sharply delineated by the stained basement membrane surrounding the tubules. The glomerular capillaries (C) and the epithelium of Bowman’s capsule (BC) also show PAS-positive basement membranes. Х360.

 

The Feulgen reaction is based on the cleavage of purines from the deoxyribose of DNA by mild acid hydrolysis; the sugar ring then opens with the formation of aldehyde groups.

Again, the newly formed aldehyde groups react with the Schiff reagent to give the distinctive magenta color. The reaction of the Schiff reagent with DNA is stoichiometric, meaning that the product of this reaction is measurable and proportional to the amount of DNA. It can be used, therefore, in spectrophotometric methods to quantify the amount of DNA in the nucleus of a cell. RNA does not stain with the Schiff reagent because it lacks deoxyribose.

 

Immunocytochemistry

The specificity of a reaction between an antigen and an antibody is the underlying basis of immunocytochemistry. Antibodies, also known as immunoglobulins, are glycoproteins that are produced by specific cells of the immune system in response to a foreign protein, or antigen. In the laboratory, antibodies can be purified from the blood and conjugated (attached) to a fluorescent dye. In general, fluorescent dyes (fluorochromes) are chemicals that absorb light of different wavelengths (e.g., ultraviolet light) and then emit visible light of a specific wavelength (e.g., green, yellow, red). Fluorescein, the most commonly used dye, absorbs ultraviolet light and emits green light. Antibodies conjugated with fluorescein can be applied to sections of lightly fixed or frozen tissues on glass slides to localize an antigen in cells and tissues. The reaction of antibody with antigen can then be examined and photographed with a fluorescence microscope or confocal microscope that produces a three-dimensional reconstruction of the examined tissue.

Two types of antibodies are used in immunocytochemistry: polyclonal antibodies that are produced by immunized animals and monoclonal antibodies that are produced by immortalized (continuously replicating) antibody-producing cell lines.

In a typical procedure, a specific protein, such as actin, is isolated from a muscle cell of one species, such as a rat, and injected into the circulation of another species, such as a rabbit.

In the immunized rabbit, the rat’s actin molecules are recognized by the rabbit immune system as a foreign antigen. This recognition triggers a cascade of immunologic reactions involving multiple groups (clones) of immune cells called B–lymphocytes. The cloning of B lymphocytes eventually leads to the production of anti-actin antibodies. Collectively, these polyclonal antibodies represent mixtures of different antibodies produced by many clones of B-lymphocytes that each recognize different regions of the actin molecule. The antibodies are then removed from the blood, purified, and conjugated with a fluorescent dye. They caow be used to locate actin molecules in rat tissues or cells. If actin is present in a cell or tissue, such as a fibroblast in connective tissue, then the fluorescein-labeled antibody binds to it and the reaction is visualized by fluorescence microscopy.

Monoclonal antibodies are those produced by an antibody-producing cell line consisting of a single group (clone) of identical B-lymphocytes. The single clone that becomes a cell line is obtained from an individual with multiple myeloma, a tumor derived from a single antibodyproducing plasma cell. Individuals with multiple myelomas produce a large population of identical, homogeneous antibodies with an identical specificity against an antigen. To produce monoclonal antibodies against a specific antigen, a mouse or rat is immunized with that antigen. The activated B-lymphocytes are then isolated from the lymphatic tissue (spleen or lymph nodes) of the animal and fused with the myeloma cell line. This fusion produces a hybridoma, an immortalized individual antibody-secreting cell line. To obtain monoclonal antibodies against rat actin molecules, for example, the B-lymphocytes from the lymphatic organs of immunized rabbits must be fused with myeloma cells.

Both direct and indirect immunocytochemical methods are used to locate a target antigen in cells and tissues. The oldest immunocytochemistry technique used for identifying the distribution of an antigen within cells and tissues is known as direct immunofluorescence. This technique uses a fluorochrome-labeled primary antibody (either polyclonal or monoclonal) that reacts with the antigen within the sample. As a one-step procedure, this method involves only a single labeled antibody. Visualization of structures is not ideal because of the low intensity of the signal emission. Direct immunofluorescence methods are now being replaced by the indirect method because of suboptimal sensitivity.

Indirect immunofluorescence provides much greater sensitivity than direct methods and is often referred to as the “sandwich” or “double-layer technique.” Instead of conjugating a fluorochrome with a specific (primary) antibody directed against the antigen of interest (e.g., a rat actin molecule), the fluorochrome is conjugated with a secondary antibody directed against rat primary antibody (i.e., goat anti-rat antibody. Therefore, when the fluorescein is conjugated directly with the specific primary antibody, the method is direct; when fluorescein is conjugated with a secondary antibody, the method is indirect. The indirect method considerably enhances the fluorescence signal emission from the tissue. An additional advantage of the indirect labeling method is that a single secondary antibody can be used to localize the tissue-specific binding of several different primary antibodies. For microscopic studies, the secondary antibody can be conjugated with different fluorescent dyes so that multiple labels can be shown in the same tissue section. Drawbacks of indirect immunofluorescence are that it is expensive, labor intensive, and not easily adapted to automated procedures.

It is also possible to conjugate polyclonal or monoclonal antibodies with other substances, such as enzymes (e.g., horseradish peroxidase), that convert colorless substrates into an insoluble product of a specific color that precipitates at the site of the enzymatic reaction. The staining that results from this immunoperoxidase method can be observed in the light microscope with either direct or indirect immunocytochemical methods. In another variation, colloidal gold or ferritin (an iron-containing molecule) can be attached to the antibody molecule. These electron-dense markers can be visualized directly with the electron microscope.

 

 

Direct method of immunocytochemistry. (1) Immunoglobulin molecule (Ig). (2) Production of a polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit Igs are produced against protein x. (3) Labeling the antibody. The rabbit Igs are tagged with a label. (4) Immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x.

 

Indirect method of immunocytochemistry. (1) Production of primary polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit immunoglobulins (Ig) are produced against protein x. (2) Production of secondary antibody. Ig from a nonimmune rabbit is injected into a goat. Goat Igs against rabbit Ig are produced. The goat Igs are then isolated and tagged with a label. (3) First step of immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x.

 

 

 

Photomicrograph of a section of small intestine in which an antibody against the enzyme lysozyme was applied to demonstrate lysosomes in macrophages and Paneth cells. The brown color results from the reaction done to show peroxidase, which was linked to the secondary antibody. Nuclei counterstained with hematoxylin. Medium magnification.

 

Histologic dyes

1.     Nuclear or alcaline: hematoxylin, carmine, saphranin.

2.     Cytoplasmic or acidic: eosin, acid fushcin, picrin acid, orange.

3.     Special dyes: orsein, sudan, osmic acid.

4.     Heavy metal impragnation: silver, gold.

 

 

 

Special equipment is used for the slides preparation. E.g. microtome for sectioning resin- and paraffin-embedded tissues for light microscopy. Rotation of the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance, generally between 1 and 10 micrometers. After each forward move, the tissue block passes over the knife edge, which cuts the sections.

 

Schematic drawing of a light microscope showing its main components and the pathway of light from the substage lamp to the eye of the observer.

 

Rules of light microscopy

1.     Put a microscope on your working place.

2.     Arrange the objective of a low magnification opposite to the object-table opening at the distance 1,5 cm away.

3.     Light up the visual field using a mirror.

4.     Put a specimen on the microscope table with coverslip up. Place the section opposite to microscope frontal lens.

5.     Watching from the side, move the microscope tubus down, leave a minimal lumen between the objective and the specimen (up to 1 cm).

6.     Look through the objective and slowly rise the tubus up by means of macroscrew until the appearance of the object under observation.

7.     Observe the whole specimen while observing it under a low magnification.

8.     Choose the most particular portion while studying the specimen under a high magnification.

9.     Look through the objective and raise slowly the tubus by means of microscrew until the appearance of image.

10. Raise the tubus by turning the macroscrew, remove the specimen from the object-table.

Main rules of specimen investigation in light microscope

It is necessary to watch the specimen under the low magnification that gives the possibility to learn its general structure. Profound investigation of the specimens is one of the microstructure observation stages and its aim is to fasten the shape and disposition of the investigated structures in a student’s memory.

Illustration includes an attentive studying of a specimen and its painting in detail.

Notice, colour and scale reproduction of histologic structures is of great importance. It is performed with colour pencils in an album with corresponding indications.

 

Principle of confocal microscopy. While a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the blind. Thus, only one very thin plane of the specimen is focused at a time.

 

Polarized light microscopy. A small piece of rat mesentery was stained with the picrosirius method, which stains collagen fibers. The mesentery was then placed on the slide and observed by transparency. Under polarized light, collagen fibers exhibit intense birefringence and appear brilliant or yellow. Medium magnification.

 

Practical arrangement of a confocal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pinhole and a detector. Light from components of the specimen that are above or below the focused plane are blocked by the blind. The laser scans the specimen so that a larger area of the specimen can be observed.

Cell fractionation allows the isolation of cell constituents by differential centrifugation. The drawings at right show the cellular organelles at the bottom of each tube after centrifugation. Centrifugal force is expressed by g, which is equivalent to the force of gravity. (1) A fragment of tissue is minced with razor blades or scissors and dissociated with a homogenizer or by ultrasound. (2) The dissociated tissue is left standing for about 20 min. Clumps of cells and fibers of extracellular matrix precipitate to the bottom. (3) The supernatant is centrifuged at 1000 g for 20 min. Nuclei precipitate. (4) The supernatant is centrifuged at 10,000 g for 20 min. Mitochondria and lysosomes precipitate. (5) The supernatant is centrifuged at 105,000 g for 120 min. Microsomes precipitate. (6) If the supernatant is first treated with sodium deoxycholate and then centrifuged at 105,000 g for 120 min, the microsomes dissociate and precipitate separately as endoplasmic reticulum membranes and ribosomes

 

 

Autoradiographs from the submandibular gland of a mouse injected with ³H-fucose 8 h before being killed. Top: With a light microscope it is possible to observe black silver grains indicating radioactive regions in the cells. Most radioactivity is in the granules of the cells of the granular ducts of the gland. High magnification. Bottom: The same tissue prepared for electron-microscope autoradiography. The silver grains in this enlargement appear as coiled structures localized mainly over the granules (G) and in the gland lumen (L). High magnification.

 

 

Autoradiographs of tissue sections from a mouse that was injected with ³H-thymidine. A: Because the autoradiographs were exposed for a very long time, the radioactive nuclei became heavily labeled and appear covered by clouds of dark granules (arrowheads). High magnification. B: Many cells were dividing at the base of the intestinal glands (arrowheads), but no cells were dividing in the pancreas (long arrow). Low magnification. C: A section of a lymph node shows that cell division occurs mostly at the germinal centers of this structure (arrow). Low magnification.

Photomicrograph of a rat kidney section treated by the Gomori method to demonstrate the enzyme alkaline phosphatase. The sites where this enzyme is present (cell surface) stain intensely with black (arrows). Medium magnification.

Photomicrograph of an intestinal villus stained by PAS. Staining is intense in the cell surface brush border (arrows) and in the secretory product of goblet cells (G) because of their high content of polysaccharides. The counterstain was hematoxylin. High magnification.

 

 

 

Compounds that have affinity toward another molecule can be tagged with a label and used to identify that molecule. (1) Molecule A has a high and specific affinity toward a portion of molecule B. (2) When A and B are mixed, A binds to the portion of B it recognizes. (3) Molecule A may be tagged with a label that can be visualized with a light or electron microscope. The label can be a fluorescent compound, an enzyme such as peroxidase, a gold particle, or a radioactive atom. (4) If molecule B is present in a cell or extracellular matrix that is incubated with labeled molecule A, molecule B can be detected.

 

 

 

Ultracentrifugation (A) and chromatography (B): methods of protein isolation. A: A mixture of proteins obtained from homogenized cells or tissues is submitted to centrifugation at high speed for several hours. The proteins separate into several bands, depending on the size and density of the protein molecules. The ultracentrifugation medium is drained and collected in several fractions that contain different proteins, which can be analyzed further. B: A solution containing a mixture of proteins obtained from homogenized cells or tissues is added to a column filled with particles that have different chemical properties. For instance, the particles may have different electrostatic charges (attracting proteins according to their charge) or different sizes of pores (acting as sieves for different-sized molecules). As the proteins migrate through the column, their movement is slowed according to their interaction with the particles. When the effluent is recovered, the different groups of proteins may be collected separately.

Gel electrophoresis: a method of protein isolation. A: Isolation of proteins. (1) Mixtures of proteins are obtained from homogenized cells or tissues. They are usually treated with a strong detergent (sodium dodecyl sulfate) and with mercaptoethanol to unfold and separate the protein subunits. (2) The samples are put on top of a slab of polyacrylamide gel, which is submitted to an electrical field. The proteins migrate along the gel according to their size and shape. (3) A mixture of proteins of known molecular mass is added to the gel as a reference to identify the molecular mass of the other proteins. B: Detection and identification of the proteins. (1) Staining. All proteins will stain the same color. The color intensity is proportional to the protein concentration. (2) Autoradiography. Radioactive proteins can be detected by autoradiography. An x-ray film is apposed to the gel for a certain time and then developed. Radioactive proteins will appear as dark bands in the film. (3) Immunoblotting. The proteins can be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with an antibody made against proteins that may be present in the sample.

Photomicrograph of a mouse decidual cell grown in vitro. The protein desmin, which forms intermediate filaments, was detected with an indirect immunofluorescence technique. A mesh of fluorescent intermediate filaments occupies most of the cytoplasm. The nucleus (N) is stained blue. High magnification

 

 

Electron micrograph showing a section of a pancreatic acinar cell that was incubated with anti-amylase antibody and stained by protein A coupled with gold particles. Protein A has high affinity toward antibody molecules. The gold particles appear as very small black dots over the mature secretory granules and the forming granules in the Golgi complex.

 

 

How different 3-dimensional structures may appear when thin-sectioned. A: Different sections through a hollow ball and a hollow tube. B: A section through a single coiled tube may appear as sections of many separate tubes. C: Sections through a solid ball (above) and sections through a solid cylinder (below).

 

Main steps in preparing tissue specimens for an electron microscopy

1.     Selection and chemical fixation of the material for an electron microscopy (glutaraldehyde and osmium tetroxide)

2.     Dehidration (substitution) and infiltration (ethyl alcohol, acetone)

3. Clearing

2.     Embedding (in plastics and epoxy resins)

3.     Sectioning with ultramicrotome (0,02-0,1 mkm thick)

4.     Staining (contrasting) with heavy metal solt (lead citrate, uranilacetate)

 

Photograph of the JEM-1230 transmission electron microscope. (Courtesy of JEOL USA, Inc., Peabody, MA.)

Schematic view of a transmission electron microscope with its lenses and the pathway of the electrons. CCD, charged coupled device.

 

Electron micrographs of 3 cell fractions isolated by density gradient centrifugation. A: Mitochondrial fraction, contaminated with microsomes. B: Microsomal fraction. C: Lysosomal fraction. High magnifications. (Courtesy of P Baudhuin.)

 

Detection of acid phosphatase. Electron micrograph of a rat kidney cell showing 3 lysosomes (ly) above the nucleus (N). The dark precipitate within these structures is lead phosphate that precipitated on places where acid phosphatase was present. x25,000.

 

Superficial complex. Cell membrane

The plasma membrane is a lipid-bilayered structure visible with transmission electron microscopy. The plasma membrane (cell membrane) is a dynamic structure that actively participates in many physiologic and biochemical activities essential to cell function and survival. When the plasma membrane is properly fixed, sectioned, stained, and viewed on edge with the transmission electron microscope (TEM), it appears as two electron-dense layers separated by an intermediate, electron-lucent (nonstaining) layer.

The total thickness of the plasma membrane is about 8 to 10 nm. The plasma membrane is composed of an amphipathic lipid layer containing embedded integral membrane proteins with peripheral membrane proteins attached to its surfaces. The current interpretation of the molecular organization of the plasma membrane is referred to as the modified fluid–mosaic model.

Diagram of a plasma membrane showing the modified fluid–mosaic model. The plasma membrane is a lipid bilayer consisting primarily of phospholipid molecules, cholesterol, and protein molecules. The hydrophobic fatty-acid chains of phospholipids face each other to form the inner portion of the membrane, whereas the hydrophilic polar heads of the phospholipids form the extracellular and intracellular surfaces of the membrane. Cholesterol molecules are incorporated within the gaps between phospholipids equally on both sides of the membrane. Note the elevated area of the lipid raft that is characterized by the high concentration of glycosphingolipids and cholesterol. It contains large numbers of integral and peripheral membrane proteins. The raft protrudes above the level of asymmetrically distributed phospholipids in the membrane bilayer (indicated by the different colors of the phospholipid heads). Carbohydrate chains attach to both integral and peripheral membrane proteins to form glycoproteins, as well as to polar phospholipid heads to form glycolipids.

 

The membrane consists primarily of phospholipid, cholesterol, and protein molecules. The lipid molecules form a lipid bilayer with an amphipathic character (it is both hydrophobic and hydrophilic). The fatty-acid chains of the lipid molecules face each other, making the inner portion of the membrane hydrophobic (i.e., having no affinity for water). The surfaces of the membrane are formed by the polar head groups of the lipid molecules, thereby making the surfaces hydrophilic (i.e., they have an affinity for water). Lipids are distributed asymmetrically between the inner and outer leaflets of the lipid bilayer, and their composition varies considerably among different biologic membranes.

In most plasma membranes, protein molecules constitute approximately half of the total membrane mass. Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely. These proteins are called integral membrane proteins. The other types of protein — peripheral membrane proteins — are not embedded within the lipid bilayer. They are associated with the plasma membrane by strong ionic interactions, mainly with integral proteins on both the extracellular and intracellular surfaces of the membrane. In addition, on the extracellular surface of the plasma membrane, carbohydrates may be attached to proteins, thereby forming glycoproteins; or to lipids of the bilayer, thereby forming glycolipids. These surface molecules constitute a layer at the surface of the cell, referred to as the cell coat or glycocalyx. They help establish extracellular microenvironments at the membrane surface that have specific functions in metabolism, cell recognition, and cell association and serve as receptor sites for hormones.

So, the main biochemical components of plasma membrane are:

Lipids. Lipids are present in cell membranes as phospholipids, sphingolipids, and cholesterol. Each phospholipid molecule has a polar (hydrophilic) phosphate-containing head group and a nonpolar (hydrophobic) pair of fatty-acid tails. Membrane phospholipids are arranged in a bilayer with their tails directed toward one another at the center of the membrane. In electron micrographs of osmium-stained tissue, a single membrane, or unit membrane, has 2 dark outer lines with a lighter layer between them.

Cholesterol is amphipatic and becomes intercalated between phospholipids in membranes. It increases the stability of the bilayers and prevents the loss of membrane liquidity at low temperatures. The concentration of phospholipids and cholesterol varies in the membranes of organisms that live at temperature extremes. Presumably, this variation maintains membrane fluidity above crucial threshold levels. If membrane fluidity falls below these hypothetical thresholds, vital functions such as selective membrane transport may cease and cell will die.

Protein. Protein may comprise over 50 % of membrane weight. Most membrane proteins are globular and belong to one of the following 2 groups: a. Integral membrane proteins are tightly lodged in the lipid bilayer; detergents are required to extract them. They are folded, with their hydrophilic amino acids in contact with the phosphate groups of the membrane phospholipids and their hydrophobic amino acids in contact with the fatty-acid tails. Some protrude from onlyone membrane surface, while others, called transmembrane proteins, penetrate the entire membrane and protrude from both sides.

b. Peripheral membrane proteins are more loosely associated with the inner or outer membrane surface; some are globular, some filamentous.

Carbohydrates. Carbohydrates occur on plasma membranes mainly as oligosaccharide moieties of membrane glycoproteins and glycolipids. Membrane oligosaccharides have a characteristic branching structure and project from the cell’s outer surface, forming a superficial coat called the glycocalyx that participates in cell adhesion and recognition.

 

Microdomains of the plasma membrane, known as lipid rafts, control the movement and distribution of proteins within the lipid bilayer. The fluidity of the plasma membrane is not revealed in static electron micrographs. Experiments reveal that the membrane behaves as though it were a two-dimensional lipid fluid. For many years it was thought that integral membrane proteins moved freely within the plane of the membrane; this movement was compared to the movement of icebergs floating in the ocean. However, recent evidence shows that the distribution and movement of proteins within the lipid bilayer is not as random as once thought. Localized regions within the plasma membrane contain high concentrations of cholesterol and glycosphingolipids. These regions are called lipid rafts. Owing to the high concentration of cholesterol and the presence of longer, highly saturated fatty-acid chains, the lipid raft area is thicker and exhibits less fluidity than the surrounding plasma membrane. Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be viewed as “signaling platforms” floating in the ocean of lipids. Each individual raft is equipped with all of the necessary elements (receptors, coupling factors, effector enzymes, and substrates) to receive and convey specific signals. Signal transduction in lipid rafts occurs more rapidly and efficiently because of the close proximity of interacting proteins. In addition, different signaling rafts allow for the separation of specific signaling molecules from each other.

Integral membrane proteins can be visualized with the special tissue preparation technique of freeze fracture. The existence of protein within the substance of the plasma membrane (i.e., integral proteins) was confirmed by a technique called freeze fracture. When tissue is prepared for electron microscopy by the freeze fracture process, membranes typically split or cleave along the hydrophobic plane (i.e., between the two lipid layers) to expose two interior faces of the membrane, an E-face and a P-face. The E-face is backed by extracellular space, whereas the P-face is backed by cytoplasm ( protoplasm). The numerous particles seen on the E- and P-faces with the TEM represent the integral proteins of the membrane. Usually, the P-face displays more particles, thus more protein, than the E-face.

 

Freeze fracture examination of the plasma membrane. a. View of the plasma membrane seen on edge, with arrow indicating the preferential plane of splitting of the lipid bilayer through the hydrophobic portion of the membrane. When the membrane splits, some proteins are carried with the outer leaflet, though most are retained within the inner leaflet. b. View of the plasma membrane with the leaflets separating along the cleavage plane. The surfaces of the cleaved membrane are coated, forming replicas; the replicas are separated from the tissue and examined with the TEM. Proteins appear as bumps. The replica of the inner leaflet is called the P-face; it is backed by cytoplasm (protoplasm). A view of the outer leaflet is called the E-face; it is backed by extracellular space. c. Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P-face of the membrane of the adjoining cell. The cleavage plane has jumped from the membrane of one cell to the membrane of the other cell, as indicated by the clear space (intercellular space) across the middle of the figure. Note the paucity of particles in the E-face compared with the P-face, from which the majority of the integral membrane proteins project.

 

Integral membrane proteins have important functions in cell metabolism, regulation, and integration. Six broad categories of membrane proteins have been defined in terms of their function: pumps, channels, receptors, linkers, enzymes, and structural proteins.

Different functions of integral membrane proteins. The six major categories of integral membrane proteins are shown in this diagram: pumps, channels, receptors, linkers, enzymes, and structural proteins. These categories are not mutually exclusive. A structural membrane protein involved in cell-to-cell junctions might simultaneously serve as a receptor, enzyme, linker, or a combination of these functions. The categories are not mutually exclusive (e.g., a structural membrane protein may simultaneously serve as a receptor, an enzyme, a pump, or any combination of these functions).

 

Pumps serve to transport certain ions, such as Na⁺, actively across membranes. Pumps also transport metabolic precursors of macromolecules, such as amino acids and sugars, across membranes, either by themselves or linked to the Na⁺ pump.

Channels allow the passage of small ions, molecules, and water across the plasma membrane in either direction (i.e., passive diffusion). Gap junctions formed by aligned channels in the membranes of adjacent cells permit passage of

ions and small molecules from the cytoplasm of one cell to the cytoplasm of the adjacent cells.

Receptor proteins allow recognition and localized binding of ligands (molecules that bind to the extracellular surface of the plasma membrane) in processes such as hormonal stimulation, coated-vesicle endocytosis, and antibody reactions.

Linker proteins anchor the intracellular cytoskeleton to the extracellular matrix. Examples of linker proteins include the family of integrins that link cytoplasmic actin filaments to an extracellular matrix protein (fibronectin).

Enzymes have a variety of roles. ATPases have specific roles in ion pumping: ATP synthase is the major protein of the inner mitochondrial membrane, and digestive enzymes such as disaccharidases and dipeptidases are integral membrane

proteins.

Structural proteins are visualized by the freeze fracture method, especially where they form junctions with neighboring cells. Often, certain proteins and lipids are concentrated in localized regions of the plasma membrane to carry out specific functions. Examples of such regions can be recognized in polarized cells such as epithelial cells.

Integral membrane proteins move within the lipid bilayer of the membrane. Particles bound to the membrane can move on the surface of a cell; even integral membrane proteins, such as enzymes, may move from one cell surface to another (e.g., from apical to lateral) when barriers to flow, such as cell junctions, are disrupted. The fluidity of the membrane is a function of the types of phospholipids in the membrane and variations in their local concentrations. As previously mentioned, lipid rafts containing integral membrane proteins may move to a different region of the plasma membrane. The movement of an integral protein anchored on a lipid raft makes signaling more precise and prevents nonspecific interactions. The lateral migration of proteins is often limited by physical connections between membrane proteins and intracellular or extracellular structures. Such connections may exist between:

• proteins associated with cytoskeletal elements and portions of the membrane proteins that extend into the adjacent cytoplasm,

• the cytoplasmic domains of membrane proteins, and

• peripheral proteins associated with the extracellular matrix and the integral membrane proteins that extend from the cell surface (i.e., the extracellular domain).

Through these connections, proteins can be localized or restricted to specialized regions of the plasma membrane or act as transmembrane linkers between intracellular and extracellular filaments.

Cell injury often manifests as morphologic changes in the cell’s plasma membrane, which results in the formation of plasma-membrane blebs. These are dynamic cell protrusions of the plasma membrane that are commonly observed in acute cell injury, in dividing and dying cells, and during cell movement. Blebbing is caused by the detachment of the plasma membrane from underlying actin filaments of the cell cytoskeleton. Cytoskeletal poisons that act on actin filaments such as phalloidin and cytochalasin-B cause extensive membrane blebbing.

 

Membrane Transport and Vesicular Transport

Substances that enter or leave the cell must traverse the plasma membrane. Some substances (fat-soluble and small, uncharged molecules) cross the plasma membrane by simple diffusion down their concentration gradient. All other molecules require membrane transport proteins to provide them with individual passage across the plasma membrane.

There are generally two classes of transport proteins:

• Carrier proteins transfer small, water-soluble molecules. They are highly selective, often transporting only one type of molecule. After binding to a molecule designated for transport, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane (Fig. 2.7b). Some carrier proteins, such as the Na⁺/K⁺ pump or H⁺ pump, require energy for active transport of molecules against their concentration gradient. Other carrier proteins, such as glucose carriers, do not require energy and participate in passive transport.

• Channel proteins also transfer small, water-soluble molecules. In general, channels are made of transmembrane proteins with several membrane-spanning domains that create hydrophilic channels through the plasma membrane. Usually, channel proteins contain a pore domain that partially penetrates the membrane bilayer and serves as the ion- selectivity filter. The pore domain is responsible for exquisite ion selectivity, which is achieved by regulation of its three-dimensional structure. Channels are ion selective and are regulated on the basis of the cell’s needs. Channel protein transport can be regulated by membrane potentials (e.g., voltage-gated ion channels ieurons), neurotransmitters (e.g., ligandgated ion channels such as acetylcholine receptors in muscle cells), or mechanical stress (e.g., mechanically gated ion channels in the internal ear).

Vesicular transport maintains the integrity of the plasma membrane and also provides for the transfer of molecules between different cellular compartments.

Some substances enter and leave cells by vesicular transport, a process that involves configurational changes in the plasma membrane at localized sites and subsequent formation of vesicles from the membrane or fusion of vesicles with the membrane.

The major mechanism by which large molecules enter, leave, and move within the cell is called vesicle budding. Vesicles formed by budding from the plasma membrane of one compartment fuse with the plasma membrane of another compartment. Within the cell, this process ensures intercompartmental transfer of the vesicle contents. Vesicular transport involving the cell membrane may also be described in more specific terms:

• Endocytosis is the general term for processes of vesicular transport in which substances enter the cell.

• Exocytosis is the general term for processes of vesicular transport in which substances leave the cell.

 

Membrane Organization: The widely accepted fluid mosaic model describes biologic membranes as “protein icebergs in a lipid sea.” Integral proteins exhibit lateral mobility and may undergo rearrangement determined by their association with peripheral proteins, cytoskeletal filaments within the cell, membrane components of adjacent cells, and components of the extracellular matrix. Integral proteins not anchored by such associations sometimes diffuse to and accumulate in one membrane region, a process termed capping.

The ultrastructure and molecular organization of the cell membrane. The dark lines at left represent the two densw layers observed in the electron microscope; these are caused by deposit of osmium in the hydrophilic portions of the phospholopid molecules.

Electron micrograph of e section of the surface of an epithelial cell, showing the unit membrane with its two dark lines enclosing a clear band. The granular material on the surface of the membrane is the glycocalyx. x100,000.

 

 

Schematic drawing of the molecular structure of the plasma membrane. Note the one-pass and multipass transmembrane proteins. The drawing shows a peripheral protein in the external face of the membrane, but the proteins are present mainly in the cytoplasmic face.

 

Membrane Functions:

1. Selective permeability. The cell membrane forms an effective seal between a cell or organelle’s internal and external environment, preventing intrusion of harmful substances, dispersion of macromolecules, and dilution of enzymes and substrates. However, membranes display selective permeability, essential to maintaining the functional steady state, or homeostasis, required for cell survival. Homeostatic mechanisms attributable to the cell membrane maintain optimal intracellular concentrations of ions, water, enzymes, and substrates. Three mechanisms allow passage of selected molecules.

a. Passive diffusion. Certain substances (e.g., water) can cross the membrane in either direction, following a concentration gradient. Passive diffusion does not require energy expenditure.

b. Facilitated diffusion. Certain molecules (e.g., glucose) cannot freely diffuse across membranes but must be helped across by a membrane component. This facilitated diffusion is often unidirectional, but it follows a concentration gradient and requires no energy.

c. Active transport. Some nondiffusible molecules can move into or out of cell either along or against a concentration gradient. Such movement requires energy, usually as ATP. An example of this active transport is the sodium pump (Na⁺/K⁺-ATPase), which can expel sodium ions from a cell even when the external sodium concentration is higher than the internal one.

2. Transduction of signals. Receptors with strong binding affinities for extrinsic signals such as hormones are located at the cell surface. The signal molecule to which a receptor binds specifically is called its ligand. Once receptors bind their signal molecules, they may transmit the signal to the cell interior (a phenomenon called transduction of signals) by one of a variety of mechanisms:

a. Receptors may transmit the signal through their association with cytoskeletal components at the inner surface of the membrane.

b. Receptors may interact with other membrane components to produce second-messenger molecules, which then transmit the message to the cell’s interior.

c. Signal-receptor complexes may be moved into the region of a coated pit and hi endocytosed, carving the signal into the cell.

d. The receptor itself may have enzyme activity (stimulated by binding the signal molecule) and transmit the signal by enzymatically altering intracellular proteins

3. Endocytosis. Cells engulf extracellular substances and bring them into the cytoplasm in membrane-limited vesicles by mechanisms described collectively a endocytosis.

a. In phagocytosis (“cell eating”), the cell engulfs insoluble extracellular substances such as large macromolecules or entire bacteria. The vesicles formed are termed phagosomes.

b. In pinocytosis (“cell drinking”), the cell engulfs small amounts of intercellular fluid, which may contain a variety of solutes. Pinocytotic vesicles are usually smaller than phagosomes.

4. Exocytosis. Exocytosis removes substances from the cell. Cells use this process both for secretion and for excretion of undigested material. A membrane-limited vesicle or secretory granule fuses with the plasma membrane and releases its contents into the extracellular space, without disrupting the plasma membrane.

The proteins of the plasmalemma are synthesized in the rough endoplasmic reticulum and then transported in vesicles to the Golgi complex, where they may be modified and transferred to the cell membrane. This example shows the synthesis and transport of glycoprotein, which is an integral protein of the membrane.

Schematic representations of the endocytic pathway and membrane trafficking. Ligands, such as hormones and growth factors, bind to specific surface receptors and are internalized in pinocytotic vesicles coated with clathrin and other proteins. After the liberation of the coating molecules, the pinocytotic vesicles fuse with endosomal compartment, where the low pH causes the separation of the ligands from their receptors. Membrane with receptors is returned to the cell surface to be fused. The ligands typically are transferred to lysosomes. The cytoskeleton with motor proteins is responsible for all vesicle movements described.

 

 

Internalization of low-density lipoproteins (LDL) is important to keep the concentration of LDL in body fluids low. LDL, which is rich in cholesterol, binds with high affinity to its receptors in the cell membranes. This binding activates the formation of pinocytotic vesicles from coated pits. The vesicles soon lose their coating, which is returned to the inner surface of the plasmalemma: the uncoated vesicles fuse with endosomes. In the next step, the LDL is transferred to lysosomes for digestion and separation of their components to be utilized by the cell.

 

Cells respond to chemical signals according to the library of receptors they have. In this schematic representation, 3 cells appear with different receptors, and the extracellular environment contains several ligands which will interact with appropriate receptors. Considering that extracellular environment contains a multitude of molecules, it is important that ligands and respective receptors exhibit complementary morphology and great affinity.

 

 

Diagram illustrating how G proteins switch effectors on and off.

 

Types of intercellular junctions

Junctions between epithelial cells occur in 3 major forms: Zonulae are bandlike and completely encircle the cell; maculae are disklike and attach 2 cells at a single spot; gap junctions are similar in shape to maculae but differ in composition and function. A junctional complex (formerly called a terminal bar) is any combination of intercellular junctions close to the cell apex that looks dark in the light microscope.

1. Zonula occludens. Zonulae occludentes (tight junctions, occluding junctions) are lo­cated near the cell apex and seal off the intercellular space, allowing the epithelium to isolate certain body compartments (eg, they help keep intestinal bacteria and toxins out of the bloodstream). Their structure, best seen in freeze-fracture preparations, results from the fusion of 2 trilaminar membranes to form a pentalaminar structure (as seen in transmission electron microscopy [TEM]); this fusion may require specific “tight-junction proteins.” In some tissues, tight junctions can be disrupted by removing cal­cium ions or treating with protease.

2. Zonula adherens. Zonulae adherentes (sometimes called belt desmosomes) are usu­ally just basal to the tight junctions. The membranes of the adhering cells are typically 20-90 nm apart at a zonula adherens; the gap may be wider there than in nonjunctional areas. An electron-dense plaque containing myosin, tropomyosin, alpha actinin, and vinculin is found on the cytoplasmic surface of each of the membranes participating in the junction. Actin-containing microfilaments arising from each cell’s terminal web in­sert into the plaques and appear to stabilize the junction.

3. Macula adherens. A macula adherens, or desmosome, consists of 2 dense, granular attachment plaques composed of several proteins and borne on the cytoplasmic surfaces of the opposing cell membranes. Transverse thin electron microscopic (EM) sections show dense arrays of tonofilaments (cytokeratin intermediate filaments) that insert into the plaques or make hairpin turns and return to the cytoplasm. The gap between the attached membranes is often over 30 nm. Sometimes fibrillar or granular material (prob­ably glycoprotein) is seen as a dense central line in the intercellular space. Desmosomes, distributed in patches along the lateral membranes of most epithelial cells, form particu­larly stable attachments but do not hamper the flow of substances between the cells.

4. Gap junction. A gap junction (nexus) is a disk- or patch-shaped structure, best ap­preciated by viewing both freeze-fracture and transverse thin EM sections. The intercel­lular gap is 2 nm, and the membrane on each side contains a circular patch of connexons. Each connexon is a protein hexamer with a central 1.5-nm hydrophilic pore. The connex­ons in one membrane link with those in the other to form continuous pores that bridge the intercellular gap, allowing passage of ions and small molecules (< 800 daltons). As sites of electrotonic coupling (reduced resistance to ion flow), gap junctions are impor­tant in intercellular communication and coordination; they are found in most tissues.

 

Electron micrograph of a section of epithelial cells in the large intestine showing a junctional complex with its zonula occludens (ZO), zonula adherens (ZA), and desmosome (D). Also shown is a microvillus (MV). x80,000.

 

Section of human skin showing hemidesmosomes (H) at the epithelial–connective tissue junction. Note the anchoring fibrils (arrows) that apparently insert into the basal lamina (BL). The characteristically irregular spacing of these fibrils distinguishes them from collagen fibrils. X 54,000. (Courtesy of FM Guerra Rodrigo.) B: Section of skin showing the basal lamina (BL) and hemidesmosomes (arrows). This is a typical example of a basement membrane formed by a basal lamina and a reticular lamina (to the right of the basal lamina in this micrograph). x80,000.

Electron micrograph of a small-intestine epithelial cell after cryofracture. In the upper portion, the microvilli are fractured transversely; in the lower portion, the fracture crosses through the cytoplasm of the intestinal epithelial cell. The grooves, which actually lie in the lipid (middle) layer of each plasmalemma, reveal that the membranes of adjoining cells were fused in the zonula occludens. x100,000.

 

 

The main structures that participate in cohesion among epithelial cells. The drawing shows 3 cells from the intestinal epithelium. The cell in the middle was emptied of its contents to show the inner surface of its membrane. The zonula occludens and zonula adherens form a continuous ribbon around the cell apex, whereas the desmosomes and gap junctions make spotlike plaques. Multiple ridges form the zonula occludens, where the outer laminae of apposed membranes fuse.

 

Model of a gap junction (oblique view) depicting the structural elements that allow the exchange of nutrients and signal molecules between cells without loss of material into the intercellular space. The communicating pipes are formed by pairs of abutting particles, which are in turn composed of 6 dumbbell-shaped protein subunits that span the lipid bilayer of each cell membrane. The channel passing through the cylindrical bridges (arrow in A) is about 1.5 nm in diameter, limiting the size of the molecules that can pass through it. Fluids and tracers in the intercellular space can permeate the gap junction by flowing around the protein bridges. B: Gap junction between living cells as seen on a cryofracture preparation. The junction appears as a plaquelike agglomeration of intramembrane protein particles. x45,000. C: Gap junction between 2 rat liver cells. At the junction, 2 apposed membranes are separated by a 2-nm-wide electron-dense space, or gap. x193,000.

 

Student’s Practical Activities

Specimen 1. Round – shaped cell (oocyte in ovary). Stained with haematoxylin and eosin.

At a low magnification of the microscope one must find oocyte under ovarium sheath. It is round-shaped cell, surrounded by one layer of small follicular cells. At a high magnification one must investigate some components of the oocyte: cell membrane, nucleus and cytoplasm. Special attention should be paid to the conformity of the cell and nucleus shape.

Illustrate and indicate: 1. Plasmalemma. 2. Cytoplasm. 3. Nucleus.

Specimen 2. Skeletal muscles of the tongue (symplast).

Stained with iron haematoxylin.

At a low magnification one must find the cylindrical structures, disposed in parallel bandles. At a high magnification one must watch the muscular fibres structure. Take notice of peripherally disposed nuclei and cross striations in cytoplasm.

Illustrate and indicate: 1. Plasmalemma. 2. Cytoplasm. 3. Nuclei. 4. Cross striations.

 

Specimen 3. Intercellular substance (elastic cartilage)

Stained with orcein and haematoxylin

At a low magnification some groups of small chondrocytes are observed in the middle of specimen. These are so-called “isogenous groups” There is a lot of intercellular substance that is produced by the cells, it consists of an oxyphilic ground substance and brown coloured fibers.

Illustrate and indicate: 1. Groups of chondrocytes. 2. Intercellular substance: a) ground substance, b) fibers.

 

References:

a) main

1.     Practical classes materials from theme “Microscope. Microscopic equipment. Histologic technique. Cytology. General structure of the cell. Superficial complex” (Intranet)

2.     Lecture presentations from theme “Introduction in histology. Cytology and embryology subject and tasks” (Intranet)

3.     Stevens A. Human Histology / A. Stevens, J. Lowe. – [second edition]. –Mosby, 2000. – P. 1–14.

4.     Wheter’s Functional Histology : A Text and Colour Atlas / [Young B., Lowe J., Stevens A., Heath J.]. – Elsevier Limited, 2006. – P. 2–5, 426–429.

5.     Inderbir Singh Text book of Human Histology with colour atlas / Inderbir Singh. – [fourth edition]. – Jaypee Brothers Medical Publishers (P) LTD, 2002. – P. 5–13.

6.     Ross M. Histology : A Text and Atlas / M. Ross W.Pawlina. – [sixth edition]. – Lippincott Williams and Wilkins, 2011. – P. 1–21, 25–35.

b) additional

1.     Eroschenko V.P. Atlas of Histology with functional correlations / Eroschenko V.P. [tenth edition]. – Lippincott Williams and Wilkins, 2008. – P. 9–10, 10–13.

2.     Junqueira L. Basic Histology / L. Junqueira, J. Carneiro, R. Kelley. – [seventh edition]. – Norwalk, Connecticut : Appleton and Lange, 1992. – P. 1–26.

3.     Disk:

http://intranet.tdmu.edu.ua/data/teacher/video/hist/  

4.     Volkov K. S. Ultrastructure of cells and tissues / K. S. Volkov, N. V. Pasechko. – Ternopil : Ukrmedknyha, 1997. – P. 6–25.

http://intranet.tdmu.edu.ua/data/books/Volkov(atlas).pdf

http://en.wikipedia.org/wiki/Histology

http://www.meddean.luc.edu/LUMEN/MedEd/Histo/frames/histo_frames.html

http://www.udel.edu/biology/Wags/histopage/histopage.htm