HISTOLOGY FULL-TEXT

William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA

Chapter 30 TECHNIQUES OF EXPERIMENTAL MORPHOLOGY

The histology so far has given details of the appearance of dead cells, tissues and organs, and of their functions, but this functional knowledge cannot be derived from the microscopic examination of a single fixed specimen. Cell function is learned by applying microscopy is such experimental situations as follow.
. Going over methods of morphology conveys the idea of histology as an active science, helping solve basic and clinical problems. Unfortunately, a factual, note-style format does a disservice in not contributing to another aspect. Outlining techniques gives no feel for the worth of the results of their application as evidence for the descriptions and stories of histology.
Plausible though they may sound, hypotheses of function must, above all, satisfy the evidence, old and new, and be appraised with an ever alert and sceptical mind.

A COMPARATIVE CYTOLOGY

l Interspecies comparisons
Examples:
l The many brown fat cells in hibernating mammals suggested that brown fat is related to the hibernating state, or to exposure to cold.
2 The all-cone retinas of diurnal mammals with sharp vision, e.g., squirrels, implied that cones provide for high acuity.

2 Changes in the number of cells
Examples:
l Normal and physiological
An increase in the number of anterior pituitary alpha cells in pregnancy suggested that they secrete a substance needed by the pregnant or post-partum woman - prolactin hormone.
2 Pathological
A decrease in the number of anterior-pituitary acidophil (epsilon) cells in one kind of dwarfism, relative to those seen normally, implied that they secrete something stimulating growth - somatotrophin.

3 Changes in individual cell morphology
Examples:
l Normal and physiological
The cross-banding patterns of contracted and relaxed skeletal muscle fibres were evidence for the 'sliding-filament' theory of contraction.
2 Experimentally manipulated
Numbers of granules in rat pancreatic-islet beta cells, before and after the administration of diabetes-inducing alloxan, indicated a secretion and storage of the hormone, insulin, by these cells.

B STAINS AND METHODS FOR SPECIAL STRUCTURES

Before the influence of physiological variables on certain structures can be recognized, ways must be found of demonstrating the structures clearly.

Further detailed information on histotechnique (including TEM methods and images) can be found under "Histopathology' at E Klatt's WebPath - U. Utah

l Special staining techniques exist for many structures and materials.
l Methods have been given for blood (Chapter l7.A), bone (Chapter 7.D), and nervous tissues (Chapters 10.C.3 and 11.F.l.).
2 A need for special stains has been mentioned for epithelial cell outlines, basal laminae, ground substances, elastic and reticular fibres, osteoid, melanocytes, bile canaliculi, neurosecretion and other stored secretions, Golgi body, mitochondria, etc.
3 By and large, transmission EM has superseded the LM staining of cell organelles, but many other special stains, including ones for bacteria and other pathological things, e.g., amyloid, are still used.

2 Blood vessels within an organ can be revealed by:
l Injection of the vessels with red carmine-gelatin, which is allowed to set, before thick sections are cut for microscopy.
2 Injection of the vessels with a coloured resin that sets in, maintains, and reveals the vascular pattern, after the organic tissue is destroyed - corrosion cast method. The cast can also be viewed by SEM.
3 Injection of the vessels with a radio-opaque suspension, which makes them visible with magnified roentgenography - microangiography.
4 A capillary bed can be demontrated by immunostaining for endothelium (e.g., for CD31), and viewing the thick section with laser confocal scanning microscopy.

3 Chemical basis for structural staining
It is the chemical nature of a structure that permits it to be stained by a particular stain. Its protein and other materials contain groups which bind the staining chemical.
l Thus a protein may have active carboxyl (COO-) or phosphoric (HPO42-) groups, able to bind covalently with the basic chromophoric ions, of, for example, methylene blue (tetramethylthionine chlorhydrate).
2 The same protein, at a lower pH, may have active amine (NH3+) groups which can bind acidic stains, e.g., potassium eosinate.
3 Because of this amphoteric character of proteins, pH thus determines which stain reacts with a particular protein, and also the intensity of the staining. For instance, raising the pH increases the staining by basic stains.
4 At a pH of around 6, some proteins are acidophilic, others and the nucleic acids are basophilic. Hence the use of combined stains, with two or more ingredients to reveal more structures.
5 Even though the staining pH may be increased above 6, certain materials, e.g., haemoglobin of red blood cells and granules of eosinophil white blood cells, continue to give an acidophil reaction. Oxyphil, oxyntic, eosinophilic, and acidophilic are used synonymously for cells or components behaving in this way.
6 Mordants are used as an intermediary for some stains to effect an indirect union between tissue groups and radicals of the dye, e.g., for haematoxylin's active derivative haematein.

4 Orthochromatic and metachromatic staining
l Orthochromatic staining is usual. The structure stains with the colour of the stain employed, e.g., collagen, green with light green.
2 Metachromatic staining (Chapter 5.C.6) is seen with some materials, e.g., cartilage matrix. The dye, say toluidine blue, combines with the sulphated proteoglycan in such a way that the dye molecules aggregate, causing a colour change from blue to reddish-purple.

5 Progressive and regressive staining
l Progressive staining leaves the section in the dye until it is adequately coloured.
2 Regressive staining overstains the tissue, then the excess stain is removed or differentiated out of the section by a solvent or oxidizing agent.

Specific and selective staining
l Specific staining shows just one structure or material.
2 Selective staining preferentially stains one structure or material; others are stained, but less strongly.

7 Vital staining involves the injection of materials into a living animal to reveal, say, macrophages (Chapter 5.A.4), or newly formed bone matrix (Chapter 7.F.5). Supra-vital staining is applied to live cells held briefly in culture, see below (l.3.l).

C STAINS FOR SPECIAL MATERIALS: HISTOCHEMISTRY

The chemical properties of materials, proteins in particular, have been used to reveal structures more clearly by selective staining. Another interest lies in the distribution and concentrations of chemicals, as materials per se, being used, stored, transported, altered, and secreted by cells. The histochemical and cytochemical approaches permit the visual localization of certain substances within tissues and cells, respectively.

l Fat
l Sudan dyes dissolve in fat, preserved by frozen sectioning, and colour it. This does not involve ionic combination.
2 Osmium tetroxide forms a black complex with unsaturated fat and, at the same time, acts as a fixative.

2 Glycogen, glycoproteins and proteoglycans
l These have

              H     H                    H     H
              |     |                    |     |
            - C - - C-       or        - C - - C -    linkages that
              |     |                    |     |
              OH    OH                   OH    NH2

                                         H
                                        /
2  are oxidized by Periodic Acid to  - C    (aldehyde) groups 
                                        \\   
                                         O  

3  which restore colour to Schiff's reagent (hence PAS technique).
4 Colourless Schiff's reagent/leucofuchsin is prepared by bleaching basic fuchsin with sulphurous acid.
5 Glycogen may be distinguished from glycoconjugates by a pretreatment with saliva, whose enzyme, amylase, will destroy only glycogen.
6 PAS method also shows the glycoproteins of BLs and the glycocalyx, and some mucins.
7 For EM, ruthenium red makes some glycoconjugates electron-dense (not red) and visible.

3 Deoxyribose-nucleic acid
l DNA of the nucleus could be shown by the Feulgen reaction:
2 mild acid hydrolysis unmasks aldehyde groups of the DNA, but leaves the RNA unchanged.
3 Schiff's reagent then reveals these free aldehyde groups.
4 A control is provided by repeating the procedure after a pretreatment with deoxyribonuclease, the enzyme known to remove specifically DNA.

4 Enzymes
l Enzymes require careful preservation by various methods:

2 An enzyme acts on another material, its substrate, to cause the separation or addition of groups, e.g., phosphate ions may be separated from a phosphoric ester of glycerol by a phosphatase. 3 Localization may be imprecise because of diffusion of the enzyme or coloured marker from the site of enzymatic activity. Thus, it is better to use more than one method to see if two give the same distribution pattern for the enzyme.

4 Electron-microscopic control for cell fractions

5 Inorganic materials
l To show some of the ferric iron present, e.g., in liver, by its formation of Prussian blue with ferrocyanide in solution.
2 For calcium, see I.2.7 below.

6 Actin
l When EM shows cytoplasmic filaments 4-7 nm thick, treat a section with a solution of heavy meromyosin (HMM).
2 If the filaments are F-actin, they should bind HMM at regular intervals, and oriented away from any Z lines or densities. The filaments thus become 'decorated' with arrowheads in EM.
3 Current methods of choice are immunostaining, or the use of phalloidin which binds actin, and can be conjugated to rhodamine for fluorescent visualisation.

D FLUORESCENT TECHNIQUES

Certain substances, when exposed to ultraviolet (UV) light, emit light of longer and visible wavelengths, i.e., they fluoresce. The fluorescence microscope illuminates the section with UV light, and its areas of fluorescence are viewed through eyepieces incorporating a UV filter to protect the eyes.

l Autofluorescence
Porphyrins and vitamin A are naturally occurring autofluorescent materials of interest.

2 Induced fluorescence
. Formaldehyde converts the catecholamines to fluorescent quinoline compounds. The UV microscopy of formaldehyde-fixed sections shows the distribution of norepinephrine, for example, in the sympathetic, post-ganglionic, nerve fibres and adrenal medulla. An APUD cell (Chapter 27.G), if given a suitable precursor, should form an amine, in which a formaldehyde-induced fluorescence can be shown.

Immunofluorescent visualization involves:
l The preparation of a pure sample of the material (peptide or polysaccharide), whose distribution in the tissues to be studied.
2 Injection of this substance into a rabbit, whose plasma cells will treat it as an antigen and produce antibodies against it. Testing the rabbit serum for antibody activity. Conjugation of the serum antibody with fluorescein isothiocyanate to make its position traceable, when viewed in UV light.
3 A better way is to fuse antibody-forming and malignant mouse lymphocytes in vitro and clone them. Kohler and Milstein's procedure thus taps, in combination, the potentials of antibody specificity and tumour growth, in the making of a monoclonal antibody (MoAb).
4 Treating, with the fluorescein-labelled antibody, a section from the animal or person in which the protein of interest may be present. The antigen will combine with and hold the antibody.
5 UV microscopy of the section, after washing out the uncombined antibody, reveals the location of the antigen, i.e., the material of interest, for instance, to show that a particular peptide hormone is in only a certain type of cell, already categorized by its staining properties and EM morphology, e.g., prolactin in acidophil anterior-pituitary cells.
In practice, for stronger binding and better visualization, the method employing a tagged secondary antibody is more frequently used than just one antibody.
6 The very strong bond between avidin and biotin is the basis for other very effective means of tagging reagents for immunohistology.
7 Immunostaining, with its high specificity and sensitivity, is used in electron microscopy by conjugating the antibody not with fluorescein, but with:
.. (a) ferritin, recognizable as granules; or
.. (b) a peroxidase that, when incubated with a substrate, gives a visible reaction product;
.. (b) gold particles, which do not react, are visible, and are of standardized sizes, so that two materials can be tested for at the same time, for any co-localization, or separate distributions.

E TRACERS FOR BARRIERS

l Around the body, several structures, in and around blood capillaries, combine to act physiologically as selective barriers, e.g., the blood-brain, blood-testis, blood-aqueous humour barriers, and the renal glomerular filtration barrier.
2 To find out which of the various structures holds back which macromolecules, probe or tracer molecules of known size and molecular weight, e.g., ferritin, horseradish peroxidase, etc., are injected into the blood.
3 Ultrathin sections of the organ are viewed by EM, after a pre-treatment to form a reaction product if the tracer is enzymatic, to see at which structure, say the BL, the tracer was hindered and had to accumulate. If gold particles of known size are used, they can be seen without this reaction step.
4 Tracer studies are applied to other routes of selective transport, e.g., in glands, and sites of absorption such as the gut.

F RADIOAUTOGRAPHY AND ELECTRON RADIOAUTOGRAPHY

Early histochemistry showed the distribution of certain materials in relation to cells and tissues. Electron histochemistry localizes certain compounds to organelles, and other structures within and outside the cell. But a knowledge of distribution alone is limited in its significance. It is desirable to know what is happening over a period of time to a substance: where it is being produced, from what, by what, where it is being transported, and how it is to be used.
One method for gaining this information is radioautography/autoradiography. It uses radioactive isotopes, e.g., 14C, 3H, 35S, 125I, combined in materials that will be processed, e.g., used in synthesis, by the cells, as they perform their activities. Examples:

l Fibroblasts and collagen
l Fibroblasts use the amino acid proline to form collagen.
2 If an animal is injected with proline, having some hydrogen replaced by tritium, this labelled or tagged proline is used by the fibroblast as if it were normal proline.
3 However, the tritium emits beta radiation, i.e., electrons. The presence of this radioactive emission can be shown by its action on the silver halide of a photographic emulsion, coating the histological section as a film.
4 The emulsion is exposed for several weeks to the radioactivity, before being developed and fixed.
5 Sites of concentration of radioactive material are marked by black grains, seen as black coiled threads in the EM. (The grains lying over particular structures may be counted.)
6 Thus proline, for example, can be followed into the fibroblast and its organelles, and then later into the collagen fibres themselves, by taking tissue, e.g., wound tissue, from animals at various times after the injection of the tagged material.

2 DNA-synthesis
l Another example, with very widespread application, employs an injection of tritiated thymidine.
2 This material is used in the synthesis of the deoxyribose-nucleic acid (DNA) of the nucleus that occurs during interphase, prior to cell division.
3 Tissue specimens taken after injection show radioactivity in only the cells experiencing DNA synthesis, while the tagged thymidine was circulating in the body.
4 The cells may migrate, which is shown by a change from their previous position.
5 This method also shows how mitotically active a particular tissue is, for instance, the gut epithelial cells are very active and move up from the crypts on to the villi, before being shed in only four days.
The technique also yields valuable data on the migrations and cell kinetics involved in the development (histogenesis) of tissues.
6 This radioactive approach is being supplanted by letting cells incorporate, as the thymidine analogue, bromodeoxyuridine, which can be recognized by a monoclonal antibody (MoAb).
In either case, the method is limited, if a continuation of cell division dilutes the marker until it is indistinguishable from background.

3 RNA-synthesis
Labelled uridine is injected for RNA; inadvertently labelled DNA can be removed by DNase before putting on the emulsion.

4 Rate of utilization of material
Persistence of a labelled material at a site indicates a slow rate of turnover (the label should have a long half-life).

G SURGICAL EXTIRPATION AND TRANSPLANTATION

Followed by histological and physiological examination at the cell and tissue levels, at intervals after operation, either by serial biopsies on one animal, or by killing many operated animals in groups, at intervals of time. The kinds of information obtained after various procedures are indicated by the queries given below.

l Total extirpation of an organ
What loss of function? What recovery of function? Associated with what compensatory changes in other organs? e.g., castration results in a degranulation, then an increased activity of the pituitary gonadotroph cells.

2 Partial extirpation of an organ or tissue
Extent of regeneration of the remaining tissue? What recovery of function? e.g., regeneration of liver. What follows extirpation of the nervous supply to an organ, or the ligaturing of its arterial supply or venous drainage? e.g., study of the re-innervation of denervated muscle.

3 Transplantation of tissue or organ
Can the transplanted tissue survive in the new site? Functional value of the transplanted tissue? e.g., in endocrine research, clinical organ replacement of blood vessels, cornea, kidney, lung, etc. Reaction of the site to the transplanted tissue? e.g., problems of immunity, induction phenomena.

4 Implantation of substitute materials
Reaction of the site to the implant? e.g., the use of implants to strengthen weak or broken bones; joint replacements; plastic heart valves; and pacemakers.

5 Parabiotically paired animals
The vascular systems of two live animals are connected so that the same blood passes through both. What material or cells mediating a response to a stimulus in one animal is borne in the blood, via the connection, to evoke a response in the other?

H MICROMANIPULATIVE METHODS

l Micromanipulative techniques are used blindly:
l To puncture single nerve cells or axons with microelectrodes, recording their electrical activity.
2 To eject pharmacologically active substances in the vicinity of nerve cells, whose activity is being recorded.
3 To puncture the nephron by micropipette at various points to extract fluid for analysis.
These methods are followed by a histological check on the position of the microelectrode or pipette in relation to the cells.

2 Micromanipulative techniques are used under direct microscopic control : e.g.,
l To dissect out individual cells, e.g., for tissue culture, transplantation of nuclei, biochemical analysis, physiological measurement; to irradiate parts of a cell in tissue culture, e.g., individual chromosomes at anaphase.
2 To effect surgical repairs, e.g. microsurgery of the eye, auditory ossicles, nerves, and blood vessels.

I IN VIVO AND IN VITRO TECHNIQUES

These allow living cells, tissues, and organs to be observed by microscopy, often providing confirmation of functions deduced from the histological examination of dead specimens. For some of the sites listed, incident rather transmitted light is used.

l In vivo (in the living organism)
l Perspex observation chambers set in the rabbit's ear, the skin of the mouse's back, or in the skull.
2 Frog's foot web, amphibian tail, human nail bed, and the human skin window.
3 Visible transplantation sites, such as the anterior chamber of the eye, and the chorio-allantoic membrane of the hen's egg with a window set in the shell.
4 Natural and surgically made fistulae between a viscus and the skin.
5 Exenteration of an organ, e.g., exposing the living spleen for trans-illumination on a microscope stage, and the study of its blood flow.

2 Tissue and organ culture
(In vitro - separated from the whole organism and its many interacting factors for control.)
l Careful and aseptic extirpation of the living organ or tissue (perhaps followed by an enzymatic dissociation of the cells, if only a certain kind of cell is desired).
Cell sorters then allow one to separate cells according to cell-surface markers; or cells may be 'panned for', by coating a dish with a material to which only one type sticks.
2 The tissue to be cultured is placed on a raft or adheres to the side of a tube at or near the gas-medium interface, in an incubator held at body temperature.
3 The gas phase provides oxygen for aerobic processes and CO2 for pH regulation; the liquid medium has nutrients to maintain the cells' activities.
4 Agents added to the medium may promote cell proliferation, e.g., insulin or a growth factor.
5 A cell response to other single variables (e.g., Ca2+ in the case of parathyroid cells, male sex hormone for prostatic cells, vitamin A for bone or cartilage) may be investigated by:
.. (a) observing the live tissue,
.. (b) studying the cells by EM and LM after fixation,
.. (c) measuring biochemically what the cells themselves are contributing to the medium.
6 The reaction of the cells to bacteria, viruses, chemotherapeutic and toxic agents, and to embryonic growth and inducing factors may be examined by these methods. Examples:

7 Measurement of the cells' reactions in vitro to stimuli is possible:
3 Observation methods
l Supra-vital staining is used to reveal certain structures. Methylene blue stains nerve fibres of neurons in culture.
2 Unstained living cells can be examined undisturbed by using modified light microscopes designed to enhance the small differences in contrast between biological structures.

J MEDICAL CYTOGENETICS

Histological medical genetics uses LM to study genes, chromosomes, and the sex chromatin, revealing relations between various disease states and visually detectable chromosomal and genetic change.

l Techniques
l Chromosomes of cultured cells

2 Sex chromatin 3 FISH - fluorescence in situ hybridization
To explore the molecular basis for disease by seeing, in individual cells, whether genes, or parts of genes, have been deleted, added, or altered
Locate the particular DNA sequence o (gene) in:

(i) nuclear DNA of interphase cells
            .   .                      (ii) DNA of metaphase chromosomes
         .         .                                  .  .  .      
 DAPI-stained __     .                             .            .
      .     / o  \    .                          .               .
  nucleus->/  ^   \    .                       . \./     \./      .
      .    \    o /   .                       .  / \     / \       .
       .    \ __^/   .                        . /   \   o   o      .
         .         .                           .       /     \     .
           .     .                              .                  .
              .                                  .       \./      .
        INTERPHASE                                 .     / \    .
                                                       .   .  .  
                                                   ARRESTED METAPHASE

STEPS

1 Separately label probe DNAs
DNA - biotin - yellow FLUORESCEIN (FITC) } colours seen in DNA - digoxigenin - antibody to digox - red RHODAMINE } UV light
2 Separately denature the target DNA and probe DNAs to make them single-stranded, and able to hybridize.
3 Put probe solution onto target cells/tissue on a slide; coverslip; incubate warm for hybridization.
4 Wash off unbound probe; process slide for probe/signal detection.
5 In the fluorescence microscope, count the number of hybridization signals (coloured sites) per nucleus, or per chromosome, noting more than 2, less than two, and translocations (e.g., red probe + yellow probe, combined at a site, show as an orange spot).
The number of probes is limited only by technical constraints and colour clashes: two-colour FISH is common.
6 For metaphase chromosomes, two probes can be used simultaneously thus: 7 The regions along the short (p) and long (q) arms of each chromosome already have numbers, from the banding patterns seen with Giemsa staining after a trypsin pretreatment - G-banding. But, mutations and small deletions cannot be seen as banding changes.

2 Diseases (examples)
1 The DiGeorge syndrome results from a one-copy submicroscopic deletion on the long arm of 22 (22q11). The variable picture includes defects in thoracic, neck, and facial development. The abnormal heart and aorta call early attention to the infant. The thymus and/or parathyroids may be absent, causing an immune deficiency (infections) and/or hypocalcaemia.

2 Leukaemia (chronic myeloid/granulocytic)

3 In chronic B-lymphocytic leukaemia, some patients have a deletion of one of the alleles (13q14) of the retinoblastoma 1 gene, coding for a tumour-suppressor protein.

4 Mongolism (Down's syndrome)

5 Intersex states (genetic)

Note the general lines of analysis:
chromosome - gene - gene product - product's role - normal phenotype
(Chromosomal defect) - gene defect - absent/disruptive product - abnormal phenotype

3 Basic research
l Chromosome damage
Metaphase chromosome preparations are used to determine what doses of noxious agents, e.g., radiation and drugs, given previously to the tissue, cause breakages and other structural damage to chromosomes.

2 Chromosome markers

K ELECTRON MICROSCOPY

l Preparation of material for transmission EM
l Fixation: of small pieces of tissue, or centrifuged pellets of cellular material, by immersion (may result in a marked gradient of fixation); or (b) by intravascular perfusion of fixative (better fixation, but the pressure may cause distortions).
2 Imbed the dehydrated material in a plastic or an epoxy resin.
3 Cut the block into very thin sections, 0.03 µm/30 nm thick, on an ultramicrotome under microscopic observation.
4 The cut sections float out on water in a small trough, and are picked up on a thin carbon or plastic film, itself supported upon a copper grid, or are put directly on the grid.
5 This grid can later be placed in the specimen-holder of the microscope (Fig. 12).
6 The tissue can be treated in several ways before observation: 2 Transmission electron microscope (Fig. 12)
               

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3 Scanning electron microscope
l The tissue is fixed, carefully dehydrated, and coated under vacuum with very thin conducting layers of carbon and/or gold.
2 In the SEM (Fig. l3) the electron beam, l0 nm wide, scans the coated specimen, causing the emission of secondary electrons, the quantity of which can yield information about the nature of that area of the specimen's surface.
3 The secondary electrons pass to a charged scintillator, where their energy is changed to light, then converted to an electrical potential for more amplification, before being applied as the signal controlling the beam intensity in a cathode ray tube (CRT).
4 The electron beam of the CRT scans its fluorescent screen in synchrony with the 'scope beam, and builds up a picture of the surface of the specimen, which can be viewed on the screen or photographed.
5 The image has much greater depth than in LM, and yields a strong 3-D impression, when stereo pairs are photographed and viewed. The magnification range is wide, l0-l00 000, with l0 nm resolution. But the image is of the surface, unless the tissue was fractured, and is influenced greatly by the tilt of the specimen to the beam.
6 The beam also makes the specimen emit X-rays of wavelengths characteristic of the chemical elements in that part of the specimen. Thus, with an X-ray detector and analyser, the SEM acts as an electron-probe microanalyser, e.g., revealing the nature of inhaled particles in specific parts of the lung.

           
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Fig. 13 Scanning electron microscope

3 Atomic-force microscopy
1 Electron microscopy subjects the specimen to harsh processing. Atomic-force microscopy (AFM) yields 0.2 nm resolutions of unfixed, uncoated, and partly hydrated biological specimens, without the need for a vacuum.
Chromatin and collagen fibrils are two of many structures examined raw, and with mild treatments to enhance contrast.
2 AFM relies on the near-field interactions between a charged, sharp tip/probe and the specimen surface, very close by.

Fig. 14 Atomic-force microscopy in constant-force mode

                         visual image
                              ^
                        image | processing
                              |
          --------------  COMPUTER -----------------
         |                                          |    feedback
         |                                          |---controller--
     scan|control                                   |               |
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         |              ~     ~                                  ceramic
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probe v scans           v                           <............force 
        specimen___Oo0ooÖoOOooO0..0_____                        constant
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Alternative modes to constant-force are: constant-height, for the sample, while the deflections of the cantilever are recorded; and tapping, where the cantilever oscillates.

L VISUAL ANALYSIS OF SUBMICROSCOPIC ELEMENTS

l Polarizing microscope l Natural light vibrating in all planes is passed through a Nicol prism polarizer, from which it leaves displaying only one plane of vibration.
2 Such plane-polarized light can pass through many biological structures with its plane unaltered. These are called isotropic.
3 Others, having an internal submicroscopic structure, (i) with elements having one dimension longer than the others, and (ii) these elements in an orderly arrangement, alter the plane of light vibration, and are called birefringent or anisotropic.
4 The light leaving such anisotropic objects can be shown to have had its polarization plane altered by passing it through another polarizing filter, used as an analyser.
5 The plane of the analyser is set perpendicular to the plane of the light transmitted through the polarizer to the object. Thus all altered light resulting from the double refraction/anisotropy of the object, except one plane (the analyser's), is blocked. The anisotropic parts appear light against a dark background.
6 Orderly biological structures, showing anisotropy in polarized light, are collagen fibres, muscle fibres, bone matrix, cell membranes, crystals, etc.
Stains, e.g., picrosirius red for collagen, can be used to enhance the effect, which is useful for assessing fibrosis in liver, lung, and kidney.

2 X-ray diffraction
l Used to determine the molecular structure of crystallizable materials.
2 Bone mineral's diffraction pattern was compared to find the best match with the patterns of various calcium salts of known composition.
3 Proteins and nucleic acids that can be crystallized, e.g., from haemoglobin and DNA to histones and DNA-histone complexes, have been very fruitfully studied to relate changes in their molecular conformation with the tasks that they do. Enough is known to make sound predictions of protein molecular shape and active sites, from the amino-acid (and nucleotide) sequences. Gorgeous, fascinating, and revealing, describe the images of the anatomy of these biological molecules.

M QUANTITATIVE METHODS

Absolute numerical values, for such quantities as the number of red blood corpuscles and kidney glomeruli, the extent of the pulmonary alveolar surface, the number of fibres of a certain size in a nerve, are needed for the better understanding of physiological data, and their correlation with the elements seen histologically. How are measurements to be made of microscopically small biological structures?

l Morphometry
l Direct: e.g., maceration in acid and fixative of the testis, teasing out of the epididymis and measurement of its length; similar studies on individual smooth muscle fibres.
2 Direct by sampling: e.g., maceration of the kidney to free the glomeruli in their corpuscles, homogenization, dilution by a known factor, and counting of the number of glomeruli in samples in a counting chamber; counting of blood cells and corpuscles (Chapter l7.A).
3 Indirect by reconstruction or casts: e.g., from serial histological sections, construct a model of lung alveoli and measure their surface area; make a plastic cast of blood vessels or lung alveoli for measurement.

To have most structures sufficiently clearly visible for measurement means examination in thin histological sections, which bring in their train three difficulties:
.. (a) artefacts, such as shrinkage and compression distort absolute and relative values;
.. (b) they offer only a sample of the whole structure;
... (c) knowledge of the shape and extent of the structures in their third dimension is lost.
A mathematical analysis and specific formulae can be applied to data measured from sections to deduce values for structures, as three dimensional entities, such as volumes, surface areas, mean widths of trabeculae, etc.

2 Stereology
Is such a mathematical treatment, leading to formulae, of the relations between:

  1. three-dimensional structures, e.g., spheres of different sizes, tubes, curved membranes, and trabeculae;
  2. the images seen with various random probes into the structures, e.g., point, linear and planar (a histological section approaches a planar probe for structures, compared with whose dimensions its own thickness is small);
  3. and the various probabilities of encountering specific images with one kind of random probe.
The formulae devised can be applied to biological structures, after making some initial assumptions about their nature, e.g., that the gut is tubular, and kidney corpuscles spheroid; and also to structures that have departed from isotropy in a known, regular way.

3 Aids to measurement and quantitative analysis
l Eyepiece grids and graticules, counting chambers, stage micrometers: for direct measurements at the microscope.
2 The section's microscopic image may be projected, enlarged, on a screen for tracing on graph paper, or for tracing outlines, e.g., of cells, to be cut out and weighed.
3 The image may be recorded permanently as a light or electron micrograph, for measurements and tracings.
4 Features of observed images, micrographs or tracings can be entered, by means of a scanner or digitizing tablet, into an image-analysing computer, programmed to calculate such quantities as areas, number of intercepts, etc. - data that can, via the stereological formulae, give the 3-D quantities of interest.
5 Another permanent record is when a computer memory holds a complete microscopic image, built up from digitized picture elements. Such stored images save the specimen further EM-beam or ultraviolet exposure, and can be processed for image enhancement and reformatting, comparisons with stored images, measurements, and the incorporation of graphics.


William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159