BT Histological Methods and Epithelium

Histology vs cytology vs pathology

• Histology = study of tissues and how they are arranged into organs

• Cytology = study of cells

• Pathology = study of abnormal conditions, disease processes

General info on tissues

• Tissues are made up of cells and extracellular matrix

• Four basic types of tissues

– Epithelium

– Connective tissue

– Muscle

– Nervous tissue (not studied in BT course)

• Organs are made up of tissues

how are cells and tissues studied

microscopes

Light Microscope

based on interaction of tissue components with light

• Different types: bright field (simplest technique), phase contrast,

differential interference, polarizing, confocal, fluorescence, etc

structural components of light microscope

• Light source

• Series of lenses (Magnification is calculated by multiplying the magnifying power of all lenses (max ≈ 1500X))

• Stage with specimen slide (specimen must be thin enough for light to pass through it)

Three systems of lenses for light microscopes

– Condenser collects and focuses light

– Objective lenses magnify the image of the object and project it towards the eyepiece

– Eyepiece further magnifies the image and projects it toward the eye or a camera

Resolution

the smallest distance between two particles at which they can be seen as separate objects

• Resolution is determined by wavelength of illumination and by numerical aperture (NA)

• Maximal resolving power of light microscope is 0.2 μm

Resolution equation

R = 0.61λ/NA = 0.61λ/nsin(μ)

R = minimal resolvable distance between distinguishable points

• λ = wavelength of the illumination source

• NA = numerical aperture = n sin(μ)

– n = refractive index of the medium between the lens and the specimen

– μ = angle of light cone (< 90°)

How to minimize R for better/finer resolution

Do this by doing one or both of the following:

(1) decreasing the wavelength of the light source (λ)

(2) increasing the NA by:

-increasing the refractive index of the medium and/or

-increasing the angle between the stage and objective

General info on tissue preparation

Goal: section (slice) the tissue very thin so that light can shine through it and stain the tissue to visualize the different components

Basic steps:

• Preserve the tissue and remove mineralization

• Embed the tissue in something easy to slice

• Section the tissue

• Stain the tissue

Preparation of tissues steps (8 steps)

Fixation → Decalcification → Dehydration → Clearing → Embedding → Sectioning → Rehydration → Staining

Fixation

to preserve the structure and molecular composition (uses Formaldehyde or glutaraldehyde)

Decalcification

removal of mineralization (if necessary) to section

dehydration

removal of water from tissue (uses graded series of ethanol (50% to 100%))

Clearing

replace alcohol with a solvent that can form a solution with the embedding medium (such as Xylene)

Embedding

to provide rigidity in order to facilitate sectioning (Melted paraffin, hardens at room temperature)

Sectioning

cut into very thin (1-10 μm) slices (Microtome is the tool used to slice)

Rehydration

replace water (most stains are water-soluble)

basic idea of measurement of μm

1 μm = 1 micron = 1 micrometer = 1 millionth of a meter = 0.001 mm = 1000 nm

A red blood cell is 6-8 μm in diameter.

Most stains are one of the following

– Stains that differentiate between acidic and basic components of the cell (e.g., H&E)

– Specialized stains that differentiate the fibrous components of the extracellular matrix (e.g., trichrome stains)

– Metallic salts that form metal deposits on tissues (e.g., silver)

Common stain is H&E

– Hematoxylin = stains acidic components of a cell (DNA, RNA, ribosomes, etc.) blue

– Eosin = acidic dye that stains basic components of a cell (cytoplasm) pink

Basophilic

cell components that stain with a basic dye (acidic stuff like DNA/RNA)

– Methylene blue, toluidine blue, hematoxylin

Acidophilic

cell components that stain with an acidic dye (basic stuff)

– Acid fuchsin, orange G, eosin

Metachromatic

a tissue or cell component that stains a different color than the dye

– Dye molecules form aggregates in the presence of polyanions in the tissue; aggregates differ in color from individual molecules

Metachromasia

Toluidine blue stains non-metachromatic tissue blue, but stains granules of mast cells purple

PAS (Periodic acid-Schiff)

used to stain carbohydrate-rich molecules

– polysaccharides (e.g. glycogen), glycoproteins, glycolipids, mucins

Lipid Staining

• Many histological methods will remove lipids from cells (alcohol and clearing agents)

• Use dyes that are soluble in intracellular lipids

– Examples: Sudan series, Oil Red O

silver staining

• Structures that stain with silver are called argentophilic or argyrophilic

• Reticular fibers (type III collagen) are black

Fluorescence

when certain substances are irradiated by light of one wave-length, they emit light with a longer wavelength

Fluorescent Staining

– Stained sections are irradiated with UV light or laser, and the emission is in the visible portion of the spectrum.

– Fluorescent substances appear brilliant or colored on a dark background

Tetracycline – Antibiotic and an Example of Fluorescent Stain

Tetracycline will bind to bone during the mineralization process

• Bone: fluorescent labeling with tetracycline

• Tetracycline was given (to a living person) on two occasions, several

days apart.

• Can determine if mineralization was occurring during one or both of the labeling periods.

Immunocytochemistry

• Uses labeled antibodies to provide precise localization of molecules

– Develop an antibody against the macromolecule of interest

– Label the antibody with a dye (often a fluorescent dye)

Two methods: direct and indirect

Direct Immunocytochemistry

antibody against the macromolecule is labeled with a dye

Indirect Immunocytochemistry

antibody against the antibody against the macromolecule is labeled with a dye

– Indirect is more sensitive because multiple anti-antibodies can bind to the primary antibody

Hematoxylin staining

Blue: nucleus; acidic regions of the cytoplasm; cartilage matrix

Eosin staining

Pink: basic regions of the cytoplasm; collagen fibers

Trichrome stains (collagen)

Dark blue: nuclei

Red: muscle, keratin, cytoplasm

Light blue: mucinogen, collagen

Elastic stains (elastic fibers)

Orcein’s elastic stain: brown (elastic fibers)

Weigert’s elastic stain: blue (elastic fibers)

Wright’s and Giesma stain (used for differential staining of blood cells)

pink: erythrocytes, eosinophile granules

blue: cytoplasm of monocytes and lymphocytes

Light Microscope vs. Electron Microscope

1. Visible light vs. beam of electrons (created by heating a metal filament in a vacuum)

2. Light is deflected by glass lenses; electron beam is deflected by electromagnetic fields

3. Magnified image is viewed by eye vs. photographic plates, fluorescent screen, etc.

-Need to use different techniques for embedding and staining.

main difference between electron microscope vs light microscope

electron microscopy uses electrons instead of a light

Electron Microscope

• Electron micrographs are in black and white

• Dark areas = electron dense; light areas = electron lucent

– i.e., with TEM, dark areas are where electrons are blocked by the stain and light areas are where the electrons pass through the specimen and are detected

• Stains that block electrons are used (heavy metals like uranium, lead, gold, platinum)

Transmission electron microscopy

electron beam passes through a very thin (40-90 nm) section

Scanning electron microscopy

electron beam is scanned across the specimen; electrons react with a metal coating that was applied to the specimen and reflected or emitted electrons are detected

How to view a slide

Think about:

• light or electron microscopy?

• what magnification?

• morphology of individual cells and arrangement of cells within a tissue

• do you know tissue type(s)? part of the body? how is it sectioned?

• color/stains

Where is epithelium located?

It covers all body surfaces, lines body cavities & hollow organs, and forms glands.

Definition of Epithelium

◦ Sheet of closely apposed cells that separates a lumen (space) from an underlying tissue

◦ Or as a gland; invaginated epithelial cells.

Embryonic Origin of Epithelium

Epithelial cells mainly form from ectoderm & endoderm, but also

mesoderm → why epithelium lines body cavities and covers most body and organ surfaces.

◦ Ectoderm-skin & its appendages (hair & sweat glands), mammary glands

◦ Endoderm-liver, pancreas, gastrointestinal & respiratory tracts

◦ Mesoderm-reproductive system, blood vessels & body cavities

Four Common Features of All Epithelia

Tight: close apposition & highly cellular

Polarity: apical, lateral & basal cell surfaces

Avascular: no blood vessels but it is innervated

Renewal: capable of replacing damaged & dead cells; stem cells in basal layer

Basic Functions of Epithelium

1) Protection: a selective barrier (skin)

2) Absorption (small intestine)

3) Transportation: filtration (kidney), diffusion & excretion

4) Secretory (glandular epithelium)

5) Sensation (olfactory neuroepithelium)

6) Contractibility (myoepithelial cells)

Simple

◦ Single layer of cells that all lie on basement membrane (arrows)

◦ Further classified by the shape of the cell

Stratified

◦ Two or more cell layers between the basement membrane and the

free-surface

◦ The bottom layer of epithelial cells is the “basal cell layer”

◦ It is in contact with the basement membrane & is mitotically active

◦ Further classified by the shape of the surface layer of cell

Pseudostratified

◦ All cells are in contact with basement membrane (arrow) but not all

cells extend to the lumen

◦ Often mistaken for stratified because nuclei appear at different levels

Squamous

• flattened cell with centrally placed, bulging nucleus

• looks like a fried egg or pancake

Cuboidal

• cell height is equal to cell width

• appear polygon-shaped

Columnar

• tall, cylindrical cell with ovoid nucleus

• nucleus is usually found in the basal half to the center of each cell

Transitional

• surface cells change shape

• dome-shaped when tissue is relaxed vs. flattened when tissue is distended

• type of stratified epithelium

Epithelium classification based on number of cell layers AND shape of superficial cells

Simple epithelium: one layer of cells

Stratified epithelium: two or more cell layers

Pseudostratified epithelium: one layer with a stratified appearance

Simple Squamous Epithelium

Lung Alveoli (air sacs), Kidney Glomeruli

Lung alveoli: Lines structures where gas exchange, diffusion & rapid transport is required and reduces friction by secreting lubricating substances (also in blood and lymphatic vessels)

Simple Cuboidal Epithelium

Kidney Tubules

-Functions to promote secretion & absorption.

-Lines secretory ducts of gland and kidney tubule

Simple Columnar Epithelium

Intestine

-Functions to allow absorption, transcellular transport, mucous and enzyme secretion.

-Lines intestines, uterine tubes, and bronchioles.

Pseudostratified Columnar Epithelium

Trachea

-Functions to secrete mucous that is moved by cilia.

-Lines trachea, bronchi & most of upper respiratory tract

Simple squamous overview

Location: blood and lymphatic vessels, alveoli, lining of the heart

Function: secrets lubricating substance, allows diffusion and filtration

Simple cuboidal overview

Location: secretory ducts of small glands, kidney tubules

Function: allows secretion and absorption

Simple columnar overview

Location: bronchioles, uterine tubes, uterus - ciliated columnar; intestines - non-ciliated columnar

Function: allows absorption, secretes mucous and enzymes

Pseudostratified columnar overview

Location: trachea and bronchi, and most of the upper respiratory tract

(ciliated cells)

Function: secretes mucous which is moved with cilia

Stratified Squamous

2 forms: squamous moist and squamous dry

• Examples: epidermis, lining the esophagus, oral cavity & vaginal canal

Stratified Cuboidal

• Large ducts of the salivary & sweat glands

• Also in large ducts of mammary gland

Stratified Columnar

• Conjunctiva of the eye

• Also, in certain large excretory ducts

Transitional epithelium:

a type of stratified epithelium

◦ Lines the organs in the urinary system (urothelium)

◦ Surface cells are “dome cells” because of their bulging shape when the tissue is relaxed (can be classified as stratified cuboidal)

◦ Surface cells flatten when the tissue is stretched

Goblet cells in glandular epithelium

• mucous secreting cells

• mucin accumulates in the bulging apex

• nucleus & remaining cytoplasm form the “stem”

• commonly found in PSEUDOSTRATIFIED COLUMNAR and SIMPLE COLUMNAR epithelium

3 Epithelial Surfaces: each with own specializations

Apical Surface: Free upper surface that faces lumen

Basal Surface: bottom surface attached to basement membrane

Lateral Surface: Intercellular and faces adjacent epithelial cells to connect them for communication

Apical Specializations

Types of free surface specializations:

1.microvilli

2.cilia

3.stereocilia

4.keratinization

Microvilli in apical surfaces

◦ Finger-like projections of the plasma membrane

◦ Emanate from apical (free) surface of the epithelial cell into the lumen

◦ collectively it resembles a “brush border” with light microscopy

o “Individual microvilli are beyond light microscope resolution”

o Function: to increase inter-villin surface area and to facilitate absorption/secretion

o Common location: intestine

Cilia in apical surfaces

◦ Long, motile, hair-like structures (Longer than microvilli)

◦ Emanate from the apical cell surface

◦ Histologically it resembles “bed head” or a minion (Viewable with light

microscope)

◦ Function: To propel substances over the epithelial surface via rapid, rhythmic oscillations (“molecular brooms”) and To provide sensation (sensory epithelium)

◦ Common location: Respiratory system (trachea and primary bronchi) and Oviduct (uterine tube)

cilia structure

formed by a core of microtubules collectively called AXONEME

◦ 9 microtubule doublets + 2 central microtubules = AXONEME

Stereocilia in apical surfaces

◦ Closely related to microvilli (“stereocilia” is a misnomer because

there is ACTIN in its core like microvilli)

◦ Non-motile, finger-like projections that may branch distally

◦ Longer than microvilli & cilia

◦ Function: To increase surface area to absorb fluid and facilitate sperm movement

◦ Location: Epididymis & vas deferens (More specialized stereocilia with motion-detecting function are found in the sensory cells of inner ear)

Keratinization in apical surfaces

◦ Apical surface specialization of epithelial tissue

◦ Keratinized stratified squamous epithelium

◦ Dead, non-nucleated layer of epithelial cells (keratin layer) that is still hanging on to the living layer underneath

*Also referred to as ”squamous dry”

◦ Function: to protect from abrasion, friction & desiccation

◦ Location: Epidermis (inc. palm of the hand and sole of the foot) and Masticatory mucosal surfaces of oral cavity

Lateral Epithelium Specializations

◦ These are types of intercellular junctions

◦ Classification based on function

1. Tight or occluding junction (sealing)

2. Adhering or anchoring junction (adhesive)

a) Adherens junction

b) Desmosomes

3. Gap or channel-forming junction (communicating)

Occluding or tight junctions

◦ “Zonula Occludens” (ZO)

◦ When membranes of adjoining cells basically fuse in a band that completely encircles each cell

◦ impermeable barriers

◦ prevents flow of materials in the space between adjoining cells

◦ sealing junction like a zipper (Ziploc bag)

◦ the most apical of the lateral junctions

◦ Purpose: IMPERMEABILITY

Adhering or anchoring junctions

3 types (like Velcro)

A. Adherens junctions: “zonula adherens” (ZA) aka belt desmosome

◦ on lateral surface located below ZO

B. Desmosomes (D): “macula adherens” aka spot desmosome

◦ on lateral surface more basally located than ZA

C. Hemidesmosomes (H): “half a desmosome”

◦ on basal cell surface

2A. Adherens junction

◦ “zonula adherens” (ZA) aka belt desmosome

◦ A form of cell-to-cell adhering junction that helps resist separation of epithelial tissues (below occluding junctions)

◦ Transmembrane glycoproteins (cadherins) connect adjacent cells (like Velcro belts)

◦ Basic structure of the adhesion belt/belt desmosome:

◦ Transmembrane proteins (cadherins) are anchored to a dense plaque of proteins inside a cell

◦ Actin filaments extend from the plaque into the cytosol

2B. Desmosomes

◦ “macula adherens” aka spot desmosome

◦ Another form of cell-to-cell adhering junction that provides resistance to stretching and twisting

◦ Transmembrane glycoproteins (cadherins) connect adjacent cells (like Velcro spots)

Basic structure of the spot desmosome:

◦ Transmembrane proteins (cadherins) are anchored to a spot-shaped, dense plaque of proteins inside a cell

◦ Reinforced by intermediate filaments (keratin) that extend from the plaque into the cytosol

2C. Hemidesmosomes

◦ “Half a desmosome”

◦ A basal surface anchoring junction

◦ adheres epithelial cells to the basal lamina

◦ Adhering connections form between a cell’s intermediate filaments and the extracellular matrix

◦ The adhesion proteins are integrins

Junctional Complex:

◦ The appearance of two or more lateral junction features

Order of junctions from apical to basal:

◦ ZO = zonula occludens

◦ ZA = zonula adherens

◦ D = desmosome

Gap or channel- forming junctions

◦ Hollow cylinders (channel proteins) cross extracellular space

◦ Structure: 6 connexins join to form 1 connexon (the gap or pore junction between the cytoplasm of 2 adjacent cells)

◦ Allow direct, open connections between adjacent cells

◦ ions & small molecules pass freely

◦ electric & metabolic coupling of adjacent cells that coordinates function in a large group of cells

◦ Purpose: COMMUNICATION

◦ Found: cardiac muscle and smooth muscle (contraction in a wave-like manner)

*6 connexins = 1 connexon

Basal Specializations of epithelial

Types of basal specializations:

1.basal lamina

2.basement membrane

3.basal surface infoldings

4.hemidesmosomes

Basal lamina

◦ It is a subunit of the basement membrane (BM)

◦ Thin layer (20-100 nm) of ECM between the epithelial basal surface and underlying connective tissue

◦ also found between adjacent epithelial tissues

◦ ECM secreted by epithelial cells

◦ primarily Type IV collagen, laminin & heparan sulfate (proteoglycan)

◦ Too thin to be seen with standard light microscopy

◦ upon higher magnification it is made of 2 sub-layers

Basal Lamina Assembly

1. Type IV collagen self-assembles into a 2D network (like window screen mesh)

2. Laminins attach to integrin proteins in the basal cell membrane

3. Nidogen & heparan sulfate cross-link laminins to the type IV collagen network

• provides a 3D structure to BL

• binds epithelium to B

Basal Lamina Functions

1. Barrier: selective filtration (kidney)

2. Cell-to-cell interactions:

◦ attachment to connective tissue

◦ receptors (integrins) attach to basement membrane

3. Compartmentalization or separation

4. Define polarity

5. Tissue scaffolding: development and regeneration

Basement membrane

◦ Thicker structure than basal lamina

◦ Like BL, it is layer of ECM between the epithelial tissue & connective

tissue

◦ Composed of: basal lamina and a reticular lamina (BL + RL) or two fused basal laminae (BL + BL)

◦ Visible by light microscopy

What is the reticular lamina?

o Reticular fiber-rich ECM layer beneath the BL (Reticular fibers are secreted by connective tissue cells (fibroblasts))

o Component of BM (May be closely associated with the BL and together may form a basement membrane)

Basal surface infoldings

On the basal surface of some epithelial cells these are plasma membrane infoldings (pockets of basal cytoplasm)

◦ Function: to increase the basal surface area (improves absorption & secretion across the basal domain of the plasma membrane)

◦ Found: kidney tubules and certain intralobular salivary ducts known as striated ducts

Basal Surface and Metaplasia

◦ Metaplasia means “change in form”

◦ Replacement of one differentiated cell type with another mature differentiated cell type (the basal stem cells switch from producing one type of epithelial cell to another)

Intestinal Metaplasia: esophagus

◦ Example: Barrett’s esophagus is intestinal metaplasia in the lower

esophagus

◦ When esophageal epithelial cells transform to become more like those that line the intestines (from stratified squamous to mucus-secreting simple columnar)

◦ Most likely it is the result of chronic irritation of cells (GERD)

Squamous Metaplasia: oral cavity

Example: Pleomorphic adenoma

◦ Most common benign salivary gland tumor

◦ Extensive squamous metaplasia with keratin cyst formation

Carcinoma/Adenocarcinoma

◦ Common disease related to epithelium

◦ Carcinoma: cancer that forms in epithelial cells

◦ Adenocarcinoma: type of cancer that forms in glands

◦ Once the BM is breached, the malignant cells may enter the circulatory system and the disease metastasize

Definition of a gland

◦A structure formed by one or more epithelial cells that secrete fluid of a different composition to blood or intercellular fluid.

◦Secreted substances can be ions, secretory polypeptides or proteins, lipids or glycoproteins.

Secreted Substances of Glands

◦Proteins (pancreas)

◦Lipids (adrenal & sebaceous glands)

◦Complexes of carbohydrates & proteins (salivary glands)

◦All 3 substances (mammary glands)

◦Ions (sweat glands, low synthetic activity)