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