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HISTOLOGY
a subdiscipline of anatomy
the study of tissues
largely a visual science that relies on microscopy and other imaging modalities to reveal cell, tissue, and organ substructure
Goals of Histology
Make small and complex structures and processes observable
Understand the relationship between tissue structure and function
Establish a basis for learning histopathology, which involves the relationship between abnormal tissue structure and functional defects
Provide a basis for treating diseased and injured tissues
Histological Techniques
Direct Observation of Living Cells and Tissues
cells are studied while still alive
colorless
phase contrast microscope
amoeboid movement
phagocytic activity of blood cells
a. Exteriorization and Transillumination
organs with long pedicles can be brought outside the body and placed in a suitable moist container permitting their transillumination and direct microscopic analysis
b. Transparent Chamber Method
installation of chambers of metal glass in the flexible ear of rabbits
Cell/Tissue/Organ Culture
in vitro in glass cultivation
isolate the cells/tissues/organ fragments
bathed in plasma, salts, amino acids, vitamins
treat with enzymes or use spatula (to harvest)
cultivated in suspension or petridish
a. Cell Culture
non adherent, dividing cells are transferred from vessel to vessel (e.g. short term culture of WBC)
b. Tissue Culture
A.K.A. explant
immature tissue culture, incubated
embedded in coagulum of blood plasma + embryonic juice
cells proliferate in the zone of outgrowth
c. Organ Culture
maintenance of mature tissue or organ fragments
study direct effects of drugs or hormones on various tissue
Uses of Cell/Tissue/Organ Culture
study of normal and cancerous cells
development of new drugs
study intracellular parasites/viruses/mycoplasma/protozoa
determination of human karyotypes
detect genetic disorders
molecular biology and recombinant DNA technique
Mechanical Micromanipulation and Microdissection
instrument: Micromanipulator
moves glass needles or pipets so a single cell can be manipulated or directed
moves fixed cells while studied under SEM
e.g. granules, vacuoles, mitochondria can be moved
Use of Radiation Probes
selective staining and probing of organelle with colored radioactive dye combined with high intensities of light available from laser sources
bombarding with beams of radioisotope & UV light
Application: removal of nucleus from a cell & selective destruction of specific cell organelles
provides the opportunity for a new form of cellular microsurgery
Cinematography
motion pictures taken through the objectives of microscope
to record cell activity (e.g. movement of cells and organelles, mitosis, phagocytosis, muscle contraction, cilia movement)
Differential Centrifugation
cell fractionation
separation of homogeneous cell organelles from a heterogeneous population of cells
uses repeated centrifugation at progressively higher speeds
smaller components need greater centrifugal force to sediment it
steps: cells (disrupted) homogenate layered in sucrose centrifuge at high speed
Microincineration
tissue slices leave ash which retain fine structural details
minerals can be identified within the cells
Frozen Section Method
a piece of tissue is placed directly on a stage of special microtome with an outlet for carbon dioxide
for sectioning biopsy material
during operation for examination of cancer cell
Freeze Drying Technique
A.K.A. lyophilization/cryodessication
freezing uses lyoprotectant (polyhydroxycompounds)
steps: freezing primary drying secondary drying
tissue is frozen and dehydrated at low temperature in high vacuum
dehydration process typically used to preserve a perishable material or to make a material more convenient for transport
material is placed in a shell freezer (w/ dry ice, methanol, or liquid nitrogen)
reagents are added to make protein content of tissue insoluble
should be done below the materials eutectic pt.
a. Primary Drying
pressure is lowered and enough heat is applied to the material for the water to sublimate
about 95% of water is sublimated
pressure is controlled by partial vacuum
heat is brought by conduction or radiation
b. Secondary Drying
higher temperature than primary drying
removes unfrozen water molecules
after this step, only 1 -4% water is retained
Properties of a Freeze-Dried Product
sealed
lesser damage to substance than simple dehydration
material not shrunk or toughed
flavors and odor remain unchanged
can be reconstituted (rehydrated)
Use of Stain
to differentiate tissue elements as certain cellular elements to produce a contrast
most stains differentiate between the acid and basic components of cells
other stains differentiate the fibrous components of the extracellular matrix
some tissues can be stained by forming metal deposits on tissue (e.g. nerve cells and extracellular fibers)
routine stain: H&E
a. Basophilic Stain
stain the acidic components of cell (e.g. DNA, RNA)
hematoxylin (blue color)
b. Acidophilic Stain
stain the basic components of cell (e.g. cytoplasmic elements)
eosin (pink color)
Methods of Staining
1. Vital Staining - staining fresh on living, unfixed tissue cells
a. Intra Vital Staining - using intravenous injection of dyes
b. Supra Vital Staining - adding dyes to the medium of the cells already removed from the organism
2. Staining Of Fixed Dead Tissues - tissues killed, embedded, sectioned, stained and mounted on slides
Advanced Visualization Procedures
in elucidating functional aspects of the cells, tissues, and organs being studied
most commonly used techniques are histochemistry (and cytochemistry), immunocytochemistry, autoradiography, column chromatography, and gene analysis
use chemical reactions, enzymatic processes, and physicochemical processes that not only stain the tissue but also permit the localization of extracellular and intracellular molecules of interest
Immunohistochemistry
uses labeled antibodies to localize specific cell and tissue antigens and is the most sensitive, specific, and widely used histochemical method
can be used with LM or TEM
because many targeted antigens are proteins whose structure may be altered by fixation and clearing, frozen sections are often used
Labels Used in Immunohistochemistry
LM: fluorescent molecule or enzyme (e.g. peroxidase, alkaline phosphatase bound to antibody)
TEM: colloidal gold or ferritin iron bound to antibody
may use polyclonal or monoclonal antibodies
Polyclonal - more sensitivity by being directed against many antigenic determinants
Monoclonal - specific for one antigen, lower sensitivity
may be direct or indirect, with label directly attached to the primary antibody (which binds the molecule to be localized), or with the primary antibody being unlabeled and visualized by a labeled secondary antibody that binds specifically to the primary antibody
Enzyme Histochemistry
uses enzymes (such as acid phosphatase, dehydrogenases, and peroxidases) in specific cell organelles to localize those components specifically
because fixation and clearing inactivate enzymes, frozen sections are commonly used
sections are incubated in solutions containing substrates for the enzymes of interest and reagents that yield insoluble colored or electron-dense precipitates at sites of enzyme activity
Autoradiography
localizes in a tissue section a radioactive substance (drug, enzyme etc.) that the living cells metabolized
uses a radioactive isotopes which is integrated into the molecule that is being investigated
isotopes that are low energy B-emitters are usually best
H3 and C14 are commonly used
Procedures:
radioactive isotopes are incorporated into macromolecules (commonly tritium 3H)
the presence of the isotopes and the macromolecule is detected by thin layer of photographic emulsion
the slide is placed in the dark for several weeks and the radioactive particles emitted expose the emulsion
emulsion if developed like film and then cover slipped and viewed by light microscopy
Result: Microscopic exam displays the presence of silver grains over the regions where the isotope labeled molecule was located
Column Chromatography
involves packing a hollow column with a semipermeable material (often tiny resin or agarose beads) and applying a cell or tissue homogenate, or a centrifugal fraction of such a sample, to the top of the column
after the sample percolates into the column, solvent is added and allowed to flow through
timed fractions of the flow-through material (eluate), containing different molecules from the homogenate, are collected
release (elution) is retarded by interactions with the packing material and results in separation
Column Chromatography Types
1. Ion-exchange - charge
2. Gel-filtration - size
3. Affinity - binding affinity
Genetic Technology
different technologies for understanding the genes which have become tools of unlimited diagnostic and therapeutic power
A. Gene Splicing
Genes from one organism can be spliced into another using a technique called gene splicing. If a scientist wanted to splice human genes into a bacterial plasmid, he would first cut both DNA fragments with the same restriction enzyme (an enzyme that breaks DNA at certain base sequences, leaving "sticky ends"). He would then take the human gene he wanted to splice into the plasmid and connect them using the "sticky ends" left by the enzymes. He would probably then add strengthening enzymes to strengthen the bond between the DNA fragments. The transgenic bacterium would most likely then be allowed to divide repeatedly, and the resulting bacterial colony would then express the gene.
B. Genetic Fingerprinting
Genetic fingerprinting is a powerful forensic tool used to identify the perpetrator of a crime through traces of genetic material left at the scene. It utilizes repetitions of DNA sequences, which differ from person to person, to uniquely identify an individual. A multi-locus probe searches for multiple repetitions of several different DNA sequences. It is highly individualized, and is one of the best ways to get an unequivocal identification of a person. A single-locus probe searches for repetitions of only one specific sequence. It works with 50 times less genetic material, but is less definitive in its results. Scientists often use three or four single-locus probes to identify individuals, whereas only one multi-locus probe would work.
C. In Situ Hybridization
is a method of analyzing the tissue distribution of particular nucleotide sequences in DNA (e.g. specific genes) and RNA (e.g. specific mRNAs)
hybridization refers to the binding of complementary nucleotide sequences to one another with specificity
recombinant DNA technology permits copies of selected single-strand nucleotide sequences to be synthesized in large numbers
synthetic sequences complementary to the RNA or DNA sequence, an investigator wishes to localize, are termed probes and can be labeled with radioisotopes (e.g. 32P), biotin, or digoxigenin
radiolabeled probes are demonstrated by autoradiography
biotin-labeled probes are demonstrated with enzymes (e.g. peroxidase) or fluorochromes covalently linked to avidin, a molecule with high affinity for biotin
digoxigenin-labelled probes are demonstrated by indirect immunohistochemistry using antidigoxigenin primary antibodies
labeled probes were first used to analyze nucleic acids isolated from cell or tissue or tissue homogenates
the term in situ refers to the application of this technique to tissue sections, smears of cells, cultures, or even whole embryos
when such specimens are incubated with labeled probes, the probes bind to and reveal the distribution of their complementary sequences
FISH: Fluorescence In Situ Hybridization
D. Electrophoresis
Electrophoresis is a method of separating DNA fragments of different lengths. The DNA samples are placed in tiny "wells" at one end of an agarose gel. An electric current is then passed over the gel, separating the fragments. The DNA bands are then revealed with a radioactive probe.
E. Blotting and Electron Transfer
used to analyze molecules first separated by electrophoresis
gels used for electrophoresis, restrict the access of large native molecule, such as antibodies and large nucleic acids, to their target molecules
blotting and electron transfer remove the separated molecules from the gels and immobilize them on membranes
transfer from the gels to the membrane may be accomplished by blotting or by electric charge
in blotting techniques, molecules in the gel are carried by the flow of a buffer across the gel and through the membrane
the membranes carrying the more accessible target molecules are incubated with labeled antibodies or complementary nucleic acid sequences (probes) to reveal the positions and relative amounts of the molecules of interest
Types ff Blotting Techniques:
Western blotting - labeled antibodies to reveal the presence and amount of a specific protein on the membrane
Northern blotting - labeled probes reveal complementary RNA sequences
Southern blotting - labeled probes localize specific DNA sequences
Southern Blotting
F. PCR Amplification
PCR (polymerase chain reaction) amplification is an extremely powerful technique by which a single molecule of DNA can be amplified millions of times in a single afternoon. The technique has enormous applications fields from forensic science, where it can amplify trace DNA samples left at the scene of a crime; to archaeology, where it can show some of the genome of ancient organisms; to modern hospital testing, where the DNA in a tiny blood sample can be used for literally hundreds of genetic tests.
Types of Microscope
1. Light or Optical Microscope
a. polarizing
b. differential interference
c. phase-contrast
d. fluorescence
e. dark-field
f. bright-field
2. UV Microscope
3. Electron Microscope
a. TEM
b. SEM
LIGHT MICROSCOPY
Mechanism of Light Microscope
The principle is based on the wave nature of light rays, and the fact that light rays can be in phase (their peaks and valleys match) or out of phase
If the wave peak of light rays from one source coincides with the wave peak of light rays from another source, the rays interact to produce reinforcement (relative brightness)
However, if the wave peak from one light source coincides with the wave through from another light source, the rays interact to produce interference (relative darkness)
Light Source
usually lit by bulbs that emit white light (average 550 nm) of varying intensity
halogen bulbs with tungsten filaments emit intense white light and are commonly used in compound bright-field microscopes
Microscope Lenses (Light microscopes have glass lenses)
condenser lens collects light from the source and projects it as a cone through the specimen
objective lens mounted on rotating turret, enlarges and resolves the specimens image and projects it to the ocular lens
ocular lens further enlarges the image and projects it into the observers retina, a screen, or photographic emulsion
Magnification
increases the specimens apparent size
objective magnification X ocular magnification
ratio of image size to the actual size
Numerical Aperture
light-gathering capacity of the microscope
NA resolving power
measure of the size/angle of the cone of light delivered by the illuminating condenser lens to the object plane and of the cone of light emerging from the object
related to the width of the lens opening (aperture)
Resolution
measure of the capacity of the microscope to distinguish 2 close but distinct points
human eye : 200 m
light microscope : 0.2 m
electron microscope : 0.002 m
independent of magnification
calculated from the NA of the objective and the wavelength of illumination:
Refractive Index
measures the comparative velocity of light in different media
measure of the optical density of an object or the speed with which it is traversed by a light wave
the air between the lens and the coverslip bends some of the light projected through the specimen
using immersion oil between the coverslip and an oil immersion objective lens maintains the refractive index, thus improving resolution
Working Distance
distance between the surface of the lens and the surface of the cover glass or the specimen when in sharp focus
NA resolving power working distance
The Properties of the Microscope Objectives
Objective
Ring
Size
Lens
Mag
Function
Scanner
red
shortest
largest
lowest
locate structures
LPO
yellow
shorter
larger
lower
initial focusing, general outline
HPO
blue
longer
smaller
higher
shows details
OIO
white
longest
small
highest
examines microorganism
TYPES OF LIGHT MICROSCOPES
Bright-Field Microscope
most common tool of histology and histopathology
bright-field: entire field is illuminated by an ordinary condenser
specimens must be translucent and stained to provide contrast
Dark-Field Microscope
examines living microorganisms that are:
invisible in brightfield microscopy
do not stain easily
distorted by staining
uses a special condenser with an opaque disc that blocks light from entering the objective lens directly
specimen appears light against a bright background
Application:
detecting T. pallidum in the diagnosis of syphilis
Polarizing Microscope
detects orderly arrangement of fibrous proteins or stained linearly oriented structures of living cells in tissue culture or fixed stained preparations (e.g.)
provides information about structural arrangement at the molecular level
Modification: two filters
POLAROID: between the light source and condenser
ANALYZER: at the draw tube
Application: spindle fibers of dividing cells, banding patterns of striated muscle, mineral elements, ash residues
Phase-Contrast Microscope
visualizes differences of refractive index within cells and tissues using a condenser lens system containing an annular (ring-shaped) diaphragm
2 sets of light rays brought together form an image on the ocular lens containing areas that are relatively light (in phase) shades of gray black (out of phase)
basic tool for tissue culture
different protoplasmic constituents produce phase variations into intensity variations and thereby enables the eye to detect mere contrast between different structures
Application:
useful for the study of unstained cells, living or fixed
teaching films of mitosis usually employ dark medium phase contrast microscopy to render chromosomes and other cell organelles darker than the surrounding cytoplasm
Interference Microscope
combines optical features of phase contrast and polarizing microscopes to provide contrast in unstained material
provides a colored 3D-image
measures phase retardation induced by specimen components by relying on differences in refractive index
can be used to calculate mass of cellular components
compares the refracted light with an unimpeded reference beam and provide an electronic readout of the data
2 light beams separated by beam splitting prisms
Fluorescence Microscope
allows localization of substances labeled with fluorescing compounds (fluorochromes: fluorescein or rhodamine)
excitation filter between the light source and the specimen filters out all wavelength except that needed to stimulate the fluorochrome
barrier filter between the objective and ocular lenses protects the eyes from UV rays and projects only the emitted light
fluorochromes stimulated by UV light emit visible light
fluorochrome auramine for M. tuberculosis (glows yellow)
fluorescein isothiocyanate (FITC) for B. anthracis (apple green)
Application: most precise method of localizing specific proteins within tissues
Scanned-Probe Microscope
uses probes to closely examine the specimen surface without causing damage or modification
Application:
maps atomic and molecular shapes
characterizes magnetic and chemical properties
determines temperature
Types of Scanned Probe Microscopes:
Types
STM (Scanning Tunneling M.)
AFM (Atomic Force M.)
Uses
thin metal probe (tungsten)
metal and diamond probe
View
detailed view of molecule (DNA)
3D image
Perks
greater resolution than EM
no special preparations
Confocal Microscope
allows visualization of 3-D structures without cutting sections
uses a scanning laser beam to make a series of sharp images on a photomultiplier tube, computers to record, and then display these as a combined high resolution image
Application: 2- or 3-D images of cells for biomedical application
UV Microscope
sees beyond what a standard optical microscope can image
some materials that are transparent or clear in normal microscopes can be imaged with UV microscopes
can image microscopic samples in the visibleandthe UV region
have features that make them superior to normal visible range microscopes (special UV optics, light sources and cameras)
by using shorter wavelengths of UV light rather than longer wavelengths of visible light, higher image resolution can be obtained
Application: imaging protein crystals that are transparent in the visible range but can be easily seen at 280 nm due to the strong absorbance of certain amino acids
Electron Microscope
permits the visualization of ultrastructures, subcellular structures, and single macromolecules such as myosin
uses electromagnetic field, fluorescent screen/TV monitor, and electron beams (W filaments) instead of glass lenses
electromagnets spread and focus the electron beam
e-beam wavelength is far shorter than that of visible light
e-beam resolution is about 1000X greater than visible light
resolving power is about 200 nm
TEM resolving power is 0.2 nm providing a magnification of 150,000X
Advantages: high resolution, high magnification
Disadvantages: requires vacuum enclosed system, high voltage, mechanical stability, living tissue cannot be used, different way of specimen preparation, well-trained staff
Tissue Preparation for Light Microscopy
1. Fixation
preservation of tissue for study
kills the tissue and bacteria
coagulate or cross-link proteins, making them insoluble
common fixatives:
buffered formalin (4% formaldehyde in buffered isotonic saline)
Bouins fluid (picric acid)
Carnoys fixative
2. Dehydration and Clearing
dehydration: removal of water from the tissue and replacement with ethanol (50-70-100%)
fixative is also removed in early steps of dehydration by several washes of 50% ethanol for 2 hours each
clearing: 100% ethanol is replaced by solvent miscible with the embedding medium (xylene)
as the tissues become infiltrated, they become more transparent
typically, first mixture of 50% ethanol and 50% xylene followed by 100% xylene for an hour each
3. Infiltration (Interpenetration)
xylene is replaced by paraffin in an oven at 58-60C
tissues are infiltrated/saturated by immersion in a medium in which they are finally embedded (e.g. wax)
50:50 mixture of xylene and paraffin (30 minutes) two changes of 100% paraffin
first paraffin bath lasts for 2 hours, second bath is 3 hours to overnight; best not to exceed 5-6 hours since tissue tends to shrink in the heat
4. Embedding
tissue is oriented and embedded in a paraffin block
block is placed in ice water to solidify
5. Sectioning
small block of paraffin containing the tissue is mounted in microtome
microtome: designed to cut thin slices (thickness between 2 and 10 microns or .002 to 0.010 m)
paraffin affixed to a slide dissolve remove dissolved paraffin
6. Mounting and Staining
most tissues are colorless (need staining)
(e.g. dyeing or metallic coating of tissue components, H&E)
purpose of mounting: for protection and to make the preparation permanent
coverslip is placed over the section
PROCESSING TISSUES FOR LM AND TEM
PROCEDURE
PURPOSE
LM
TM
1. Fixation
Preserves tissue morphology by coagulating proteins; stops autolysis
Formaldehyde solution
Glutaraldehyde and osmium tetroxide
2. Dehydration
Removes water from cells and tissue
Pass thru graded ethanol series (35 100%)
3. Clearing
Enables cells and tissues to be penetrated with paraffin (LM) or plastic (TEM)
Benzene (organic solvent)
Propylene oxide (organic solvent)
4. Embedding
Penetrates cells and intracellular spaces giving tissue rigidity for sectioning
paraffin
Plastic (Epon)
5. Sectioning
Provides thin sections of cells and tissues
5-10 um on microtome
10-20 nm on ultramicrotome
6. Mounting
Provides supporting medium for viewing and handling
Glass slide
Fine wire grid
7. Rehydration
Removes paraffin so that tissue can be stained with aqueous solution
Pass from benzene 100% EtOH 35% EtOH
Pass down 100% EtOH 35% EtOH
8. Staining
Helps visualize tissue and cell components
Hematoxylin and Eosin
Uranyl acetate
9. Dehydration
Makes permanent
Pass from 35%EtOH 100%EtOH benzene and mount
Pass thru 100% EtOH and air dry. Store in dessicant.
COMMON STAINS AND THEIR AFFINITIES
APP.
TYPES
STAINS
LM
Basic dyes
Hematoxylin
Toluidine Blue
MB
Alcian Blue
Basophylic tissue components(DNA, RNA, polyanions such as sulfated glycosaminoglycans)
Acidic dyes
Eosin
Orange G
Acid Fuschin
Acidophilic tissue components (basic proteins in cytoplasm)
Lipid-soluble dyes
Oil red O
Sudan black
Long chain hydrocarbons (fats, oils, waxes)
Multicomponent histochemical reaction
Periodic Acid-Schiff (PAS) Reaction
Complex carbohydrates (glycogen, glycosaminoglycan)
Feulgens Reaction
Nuclear chromatin (DNA and associated proteins)
TEM
Heavy metal (electron dense)
Uranyl Acetate
Lead Citrate
Nonspecific; adsorb to surfaces and enhance contrast
Osmium Tetroxide
Actually a fixative,
but binds to phosphate groups of membrane phospholipids, enhancing contrast
Ruthenium Red
Polyanions; complex carbohydrates
e.g. Oligosaccharides of glycocalyx and glycosaminoglycans of the extracellular matrix