ANAT D502 – Basic Histology - Indiana University

13 Pages·2015·0.47 MB·English

by  Condon, Keith Warren

ANAT D502 – Basic Histology

Introduction to Histology & Microscopy
Revised 8.26.15

Reading assignment: Chapter 1: Methods (Ross & Pawlina); note Folders 1.1 and 1.3 (Clinical
Correlations)
Chapter 4: Tissues: Concept and Classification; note Folder 4.1 (Clinical
Correlation)

Outline:
I. Introduction to Histology
A. Why study Histology
B. Hierarchy of Anatomical Organization
C. Classification of Tissues
D. Cell concepts
i. Cell Theory
ii. Cells / Organisms as thermodynamic machine
iii. Endosymbiosis
II. Tissue processing for Light Microscopy
A. Fixation
B. Dehydration, infiltration, embedding
C. Sectioning
D. Staining
i. Histological
ii. Histochemical
iii. Molecular specific compounds
iv. Immunohistochemistry (IHC or ICC)
v. Autoradiography
E. Cave! Omnia sunt artificia! (A Cautionary Tale)
III. Light Microscopy
A. Definitions
B. Microscope Classification
C. Stereomicroscopes
D. Compound transmitted light microscopes
i. Design
ii. Computation of magnification and resolution
iii. Methods of illumination
E. Fluorescent microscopy
i. Confocal scanning microscopy
IV. Electron Microscopy
A. Transmission (TEM)
i. Staining
B. Scanning (SEM)

I. Introduction to Histology

Histology or microscopic anatomy or microanatomy is the study of the minute structures of cells, tissues
and organs in relation to their function . As such it requires microscopic methods of
study as the limit of human eye (macroscopic) is approx. 0.2 mm (see Text Box 1.4).

A. Why study Histology?

B. Hierarchy of Anatomical Organization
Organisms can be studied at multiple levels
Molecules (simple and macro)
Sub-cellular (organelles)
Cells (cytology)
Tissues (histology sensu strictu)
Organs (organology)
Organ systems
Organism
Supra-organismal: Societal / Ecological

Similarly, cells can be arranged into hierarchical, interdependent levels of organization

1) cell fundamental unit of the organism; performs all functions vital to its survival; over 300 defined
types in humans alone
e.g., goblet cells - epithelial glandular cells of digestive and respiratory tracts
goblet cells produce mucous secretion that provide lubrication of lumenal surfaces

2) tissue - collection of cells and cell products that perform the same general function
e.g., epithelial lining of the gut tract
epithelium forms selective barrier to lumenal contents

3) organ - group of tissues that collectively perform a common function
e.g., small intestine consists of collections of epithelia, connective tissue, muscle and nervous
tissue
small intestine is site of enzymatic digestion and absorption

4) organ system series of organs which are functionally interrelated
e.g., digestive system = oral cavity, pharynx, esophagus, stomach, small and large intestines,
rectum and anus
digestive system breaks down food items into simpler molecules

C. Classification of Tissues

Majority of cells can be grouped into 4 major types of tissue: epithelial, connective, muscle and nerve

1) epithelia - continuous layers of cells that (1) line surfaces and (2) form glands
2) connective tissue – cells embedded in an extra-cellular matrix of (1) fibers and ground
substance or (2) fluid
3) muscles - specialized contractile cells
4) nervous tissue - comprised of (1) cells specialized for conducting and transmitting
electrochemically mediated information (neurons) and (2) the cells which support this
activity (neuroglia or glia )

Intermediates exist
myoepithelial cells – spindle-shaped cells around salivary, sweat and mammary glands; these are
contractile (smooth muscle) epithelial cells that help express gland contents
neuroepithelial – e.g., hairs cells, taste buds, olfactory epithelium; cells which form an epithelium but
have the properties of neurons

D. Cell Concepts

i. Cell Theory

First developed by M. Schleiden (1838) and T. Schwann (1839), its modern incarnation is as follows:
1) Organisms consist of cells (and their products)
2) All cells come from preexisting cells (no spontaneous generation (abiogenesis))
3) Cells are smallest structural units that perform all living functions

ii. Cells / Organisms as thermodynamic machine

Laws of thermodynamics tells us two things:
1. Energy (including heat) cannot be created or destroyed
2. Entropy (or disorder) tends to increase

Life as a physico-chemical process and must conform to the laws of nature including thermodynamics

Puzzle of living organisms is that they are very highly ordered and are constantly creating new order as
they grow, reproduce and maintain themselves
How is this thermodynamically possible in a system undergoing increasing entropy?
Answer lies in relatively inefficiency of catabolism of energy

Implication of thermodynamics for cells / organisms

1. To remain in an ordered state, cells must constantly expend energy
This energy is stored in the covalant bonds of organic molecules and released in a process known
as cellular respiration
Cessation of this expenditure results in entropy (and subsequent death)

2. The inefficiency of cellular respiration results in a net increase in entropy (through the production of
heat) while providing sufficient energy to remain in an order state

3. (1) The need for energy and (2) the necessity of dissipating heat drives the “design” of multi-cellular
organisms and their organ systems

4. Limits of energy release by cellular respiration establishes constraints on body plans

iii. Endosymbiosis

Cell theory tells us that all organisms, both uni- and multi-cellular, consists of cells

Defining characteristics of eukaryotic cells
1. Genetic material separated by a nuclear envelope (i.e., nucleus)
2. DNA bound to histones (specific basic proteins)
3. Numerous membrane-bound organelles found in cytoplasm

Origin of membranous organelles
Some of the membranous organelles (nuclear membrane, ER) thought to originate from in-folding of
the plasma membrane
Other membranous organelles are thought to have arisen from a process known as endosymbiosis

endosymbiosis – form of symbiosis in which one symbiont lives within the body of another

The endosymbiotic hypothesis states that several key organelles of eukaryotes originated as symbioses
between separate single-cell organism
Such organelles include mitochondria, chloroplast, flagella and cilia
Evidence for the endosymbiotic origin of mitochondria includes:
(1) size (2) double-membrane, (3) DNA structure (4) DNA sequence

An animation on endosymbiosis can be found at the following URL:
http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter4/animation_-
_endosymbiosis.html

II. Tissue processing for Light Microscopy

In order to be viewed through a light microscope, most specimens undergo some sort of processing.
First, the tissue must be fixed to preserve structure and prevent decay by autolysis or bacterial
breakdown
Second the tissue is prepared for observation. If transmitted light or transmitted electron
microscopy is to be used, then the specimen has to be sectioned thin enough to pass
light or electrons; this requires embedding in a suitable solid medium for sectioning.

A. Methods of Fixation.

Physical fixation – tissue is preserved and hardened by rapid freezing; commonly used in surgical
biopsies
advantages: rapid processing, retention of fat, retention of most enzyme and protein function;
retention of epitopes (antibody binding sites; see below)
disadvantages: requires special equipment (cryotome) for sectioning; usually produces thicker
sections (8+ micrometers) resulting in loss of resolution; produces greater distortion of
tissue in cutting; tissue degrades with thawing

Chemical fixation – use of chemical formulations to stabilize cellular constituents (primarily large proteins
and nucleic acids)
advantages: thinner sections (2 – 8+ micrometers); less distortion
disadvantages: loss of many enzyme and protein functions; many antibody binding sites
(epitopes) lost

Methods of chemical fixation: Variety of fixatives available; those used in slide boxes described.
formalin – aqueous solution of formaldehyde; crosslinks proteins; poor fixative of lipids
osmium tetroxide – heavy metal that preserves and stains lipids and phospholipids (e.g.,
membranes)

B. Dehydration, clearing, infiltration, embedding

These steps replace the water within the cell with a more rigid material to permit thin sectioning; the
choice of this material (paraffin or resin) is determined by (1) the hardness of the tissue
(mineralized vs. non-mineralized) and (2) desired section thickness (harder > thinner).

Resins (organic monomers) are harder than paraffin and thus are most commonly used for mineralized
tissues or for electron microscopy (which requires thinner sections than does light
microscopy). Paraffin is the most common medium for light microscopy of non-
mineralized tissues.

Dehydration: water is removed by alcohols or other substances
Clearing: Involves an intermediate agent miscible in both alcohol and the embedding material; for
paraffin this is typically a benzene derivative (e.g., xylenes); many resins do not require
this step
Infiltration: The embedding media infiltrates the tissue, both intra- and inter-cellularly
Embedding surrounds the tissue with medium allowing it to be attached to a microtome

C. Sectioning

The instrument used to produce sections depends upon the method of fixation (frozen vs. chemical) and
desired thickness
cryotome (freezing microtome) – used for frozen tissue; typically produces sections of 8+
micrometer thickness
microtome – used for paraffin or resin embedded tissues; typically produces sections 4 – 8+
micrometer thickness
ultra-microtomes – used for resin embedded tissues; produces sections of less than 0.5 micrometer
thickness; primarily (but not exclusively) used for electron microscopy (EM)

D. Staining

Sections of even heavily pigmented material are optically transparent; thus, to render an image it is
necessary to impart contrast either through illumination (see Light Microscopy; below)
or staining.

i. Histological staining

Histological staining involves the use of dyes to render tissue optically opaque. These dyes are broadly
divisible into general and specific stains. General stains dye tissues differentially but
not specifically (e.g., all the tissues are shades of the same color). In contrast, specific
stains are more restricted in their uptake (e.g., orcein by elastic fibers). Dyes are often
used in combinations (e.g., hematoxylin and eosin)

The method of action of most dyes is incompletely understood; they often act through charge
attraction (See text Table 1.2)

acid dyes – carry a net negative charge (anionic), thus binds to positively charged tissue
(cationic)
tissues stained by acid dyes are termed acidophilic and are usually basic in nature
thus acid dyes preferentially bind to cytoplasm and extracellular fibers
e.g., eosin, analine blue

basic dyes – carry a net positive charge, thus bind to negatively charged tissue (anionic)
tissues stained by basic dyes are termed basophilic and are usually acid in nature
thus basic dyes preferentially bind to heterochromatin, nucleoli, ergastoplasm
e.g., methyl green, toluidine blue

General stains:
hematoxylin and eosin (H&E) is the most common general staining combination
hematoxylin – natural dye derived from the logwood tree
acts like a basic dye thru action of a mordant (agent that promotes binding of a dye to
material; e.g. alum for hematoxylin) to preferentially (but not exclusively) stain nuclei
eosin – a synthetic acid dye that preferentially (but not exclusively) stains cytoplasm and extra-
cellular fibers

Specific stains:

Masson’s trichrome – cocktail of three dyes; differentiates connective tissue fibers from cytoplasm
(epithelia, muscle)

osmium tetroxide – preserves and stains lipids and phospholipids (e.g., adipocytes, myelin,
membranes)

orcein – natural dye used to demonstrate elastin fibers

silver methods – used to demonstrate reticular fibers (argyrophilic; e.g., Wilder’s reticular stain); also
mineralizations (see Histochemistry (below)
periodic acid - Schiff (PAS) – used to demonstrate carbohydrates

Please view the following video which illustrates the steps described above in producing and H&E stained
paraffin section:

http://www.iupui.edu/~anatd502/histo_720P.mp4

ii. Histochemical staining

Histochemistry is used to localize and identify substances in a tissue by means of chemical reactions;
substances can be ions (calcium, iron, copper, zinc, etc.), proteins (primarily enzymes),
carbohydrates and lipds.

protein histochemistry
primarily enzymes; requires preservation of enzyme activity within section
enzymatic reaction between enzyme and its substrate is coupled to a marker compound that
generates a visible insoluble product
examples: tartrate-resistant acid phosphatase (TRAP), acetylcholinesterase (AChE), myosin
ATPase

ion histochemistry – chemical reactions that bind pigments to minerals
Perl’s technique demonstrates iron deposits (Prussian blue)
Von Kossa’s silver method demonstrates mineral (calcium and phosphate) ions

carbohydrate histochemistry
Periodic acid –Schiff (PAS) reaction demonstrates poly- and oligo-saccharides (e.g., glycogen
and glycoproteins)

lipid histochemistry
Frozen sections only; lipophilic dyes (Sudan back, Oil red O) suspended alcohol dissolve in
cellular lipid droplets

iii. Molecular specific compounds

By happenchance compounds have been discovered that bind to specific molecules; most of these
substances have to be coupled (conjugated) to marker compounds in order for the
binding to be observed (e.g, fluorescent molecules, enzymes, electron-dense particles)
although some are naturally fluorescent (e.g., DAPI)
phalloidin – derived from mushrooms; binds to actin
lectins – family of proteins or glycoproteins derived from plants; each binds to a specific
carbohydrate; e.g. wheat germ agglutinin
toxins – derived from various organisms; e.g., α-bungartoxin binds to acetylcholine receptors
DAPI – naturally fluorescent compound that binds to DNA

iv. Immunohistochemistry

Immunohistochemistry (IHC or immunocytochemistry (ICC)) is the use of antibodies (Ab) to localize
specific gene products in specimens, e.g. proteins such as type V collagen
Since antibodies are virtually transparent, detection of antibody binding (Ab) requires coupling
(conjugation) of the antibody to a marker compound; common conjugations include:
coupling to enzyme; subsequent histochemical reactions yields light visible insoluble product
coupling to a fluorescent molecule
coupling to a gold or electron dense particle visible in EM (5-30 nm in diameter)
Conjugation can be direct (to the Ab) or indirect (attached to a secondary Ab against the first Ab);
latter results in signal amplification

v. Autoradiography

In autoradiography radioactive substance are introduced into the animals or cells and incorporated into
tissue
Sections are covered with photographic emulsion (EM and LM)
Isotope decay results in visible silver deposits very near site of isotope
Of used with in situ hybridization to localize specific mRNA
Formerly used to birthdate cell by injection of tritiated thymidine; largely replaced by BrdU
immunocytochemistry

E. Cave! Omnia sunt artificia! (A Cautionary Tale)

As a result of the processes involved in producing histological sections, significant changes are made to
the tissue:
Formaliln fixation alters the conformation and position of many proteins
Dehydration through alcohols removes alcohol-soluble constituents (including lipids); removal of
water also produces a 10-25% shrinkage of the tissue; both of these processes results
in artificial spaces
Xylenes and other clearing agents further extract lipids and alter membrane permeability; also alter
optical properties
Many proteins and nucleotides are denatured by heating
Staining is often done at non-physiological pH levels
Sectioning can distort the tissue and introduce wrinkles (folds)

Thus, the environment preserved in histological sections is far removed from the physiological in vivo
condition (Cave! Omnia sunt artificia). As such, histological sections need to be
interpreted, not believed.

Finally, tissue sections are essentially two-dimensional representations of three-dimensional objects. The
appearance of an object depends upon it orientation relative to the plane of section.
Thus, different shaped objects can appear similarly shaped when sectioned; and
conversely, similarly shaped objects can appear different. Remember to see 2 but
think 3 (and sometimes 4).
III. Light Microscopy

A. Definitions

Microscope – instrument that magnifies an image to a level at which the retina can resolve
information

Resolution – ability to distinguish two closely spaced objects as separate entities

Working distance – vertical distance from front of objective to specimen

Depth of field – distance from the nearest object plane in focus to that of the farthest plane also
simultaneously in focus

Par focal – operating condition in which objects in focus at low magnification remain in focus at
higher magnification

Field of view – total area of specimen visible through the oculars; the width (diameter) of the field of
view is known as the optical diameter and can be used as a ruler

Numerical aperture – measure of light gathering ability of a lens; determinant of resolution

N.B. You are responsible for knowing the following table:

Limits of Resolution: Eye vs. Instruments

Distance between resolvable points (d min or R)
Human eye 0.2 mm
Bright-field microscope 0.2 μm
SEM 2.5 nm
TEM 1.0 nm

B. Microscope Classification
Many types of microscopes exist using a wide variety of sources of illumination: UV, X-ray, visible light,
electrons, confocal (laser), atomic force, etc.)

Types of light microscopes
Light microscopes can be classified in a variety of ways:
number of lenses: simple (1 lens) vs. compound (multiple lenses)
direction of illumination: reflected vs. transmitted
method of illumination (brightfield, phase, etc.; see below)

C. Stereomicroscopes

Stereomicroscopes are compound microscopes which render 3-dimensional images
Use reflected visible light
Provide large working distances, large fields of view, and large depth of field; however,
magnification and resolution are limited
best for dissection and or low-power work

D. Compound transmitted light microscopes

Use visible light transmitted through the specimen via multiple lenses to render an image
Disadvantages: small working distances, small fields of view, and little depth of field (flat image)
Advantages: higher magnification and resolution

i. Design

light source – typically 540 nm (green light; not optimal but cheap)
condenser – focuses beam of light at level of specimen
specimen – object to be examined
objective lens – gathers light that passes through specimen; typically several objective lenses of
different magnifications carried on the nosepiece
ocular lens – allows visualization of objective image; typically fixed magnification(e.g., x10)

ii. Computation of magnification and resolution

Total magnification (virtual (retinal) image) = objective lens magnification x ocular lens magnification

Resolution (the ability to distinguish two closely approximated objects = d min) is optically
determined by 3 factors: (1) wavelength of illuminating light, (2) numerical aperture of
the condenser lens, and (3) numerical aperture of the objective lens

In microscopy an image forms when light interacts with specimen, thus it’s the size
(wavelength; λ) of the light and the microscope’s ability to gather light (numerical
aperture; NA) that is important, not magnification

Specifically, R (resolution or d min) is proportional to λ / NA
to maximize resolution (= minimize distance) you must decrease λ or increase NA
the maximum resolution of light microscopy is approximately 0.2 micrometers under
optimal conditions (e.g. mitochondria)
section thickness also influences resolution; thicker sections have lower resolution
(Why?)

iii. Methods of Illumination

In transmitted light microscopy an image forms when light interacts with the specimen; by altering method
of illumination different interactions are achieved and different images obtained
Brightfield – most common method of illumination; specimens appear on white field of view (the
surround); contrast is low unless specimen absorbs / diffracts a lot of light, thus usually
requires stained tissue

Phase contrast – often used with unstained specimens or cells in vitro; special optics that uses
differences in refractive index in different parts of a cell and surround to enhance
contrast
differential interference microscopy (Nomarski) – modification of phase contrast that produces
a 3-dimensional image

Darkfield – portions of specimen appear bright against dark surround; only light scattered by
structures within specimen reach objective; useful to examine crystals (e.g., silver
grains of autoradiography).

Polarized light – polarizing filters placed on both sides of specimen
items which are birefringent (e.g., collagen, striated muscle) are able to rotate the plane of
polarized light and appear bright against a dark field

E. Fluorescent microscopy

Fluorescent microscopy – type of UV microscopy

UV light can be either transmitted or reflected (epiflourescent), latter more common
Requires fluorescent material within the specimen: either naturally occurring or by administration of
fluorescent labels (vital stains, lectins, antibodies)
Sample illuminated with UV light source causing fluorescent materials to emit light in visible
wavelength; field (background) is dark