TABLE OF CONTENTS

Methods and 1.
2.
Histology Methods - Microscopy
Histology Methods - Tissue Preparation
3. Histology Methods - Overview
Overview of Cells 4. Histology Methods - Practice Questions
5. Organelles - Nucleus
6. Organelles - ER, Golgi, Mitochondria
7. Organelles - Practice Questions
8. Cytoplasm - Cytoplasmic Inclusions
9. Cytoplasm - Cytoskeleton
10. Cytoplasm - Practice Questions
REVIEW OUTLINE

Histology ●
1. Microscopy
Light Microscopes

Methods ●
●
Transmission Electron Microscopes
Scanning Electron Microscopes
● Units of Measure

2. Tissue Preparation
● Fixation and Sectioning
● Histochemistry: Four Basic Molecules
● Staining: H&E
● Special Techniques

Histology Slides Licensed From © Histology Guide

Histology Methods [Link]

What is Histology?
The study of structure and
function of cells, tissues and
organs at the microscopic
level in order to understand
normal function and to
diagnose pathology.
Histology Methods [Link]

Light Microscopes (LM

Tissues are relatively colourless, for

visualization in the light microscope
they are usually stained with dyes
Histology Methods [Link]

Electron Microscopes (EM

Transmission EM
Electrons pass through a section of the
specimen and are absorbed at different
rates by variations in density of the tissue

1 μm
Histology Methods [Link]

Electron Microscopes (EM

Scanning EM
Electrons are reflected from the
surface of a specimen and collected
to form image of sample surface

2 μm
 Histology Methods [Link]

Units of Measure in Microscopy

These measurements can conveniently be used
as “yard sticks” to judge magnification in
micrographs

Resolution
Resolution is the smallest distance
between two points that are
discernable as separate objects

Distance between resolvable points

Human Eye 0.2 mm
Bright-field Microscope 0.2 μm
SEM 2.5 nm
TEM 1.0 nm
Histology Methods [Link]

Electron Microscope (EM Light Microscope (LM

 Histology Methods [Link]

Preparation of Tissues
1. Fixation 2. Embedding 3. Sectioning 4. Staining
Terminates metabolic Dehydration and Sliced into thin, Chemical dyes are
cell processes, prevents enclosing of tissue in transparent sections applied to stain
degradation, chemically a hard medium for using a knife mounted components with
cross-links proteins. sectioning. Paraffin on a microtome. different colors.
Formaldehyde, 70% Wax, Epon Resin)
Ethanol)

[Link] [Link]
Histology Methods [Link]

Four Basic Molecules

 Histology Methods [Link]

Basic Dyes: Hematoxylin

Basophilic structures are stained
by basic dyes

Basophilic =

Hematoxylin
 Histology Methods [Link]

Acid Dyes: Eosin

Acidophilic structures are stained
by acidic dyes

Acidophilic/Eosinophilic =

Eosin
Histology Methods [Link]

Hematoxylin and Eosin: H&E

 Histology Methods [Link]

Low Magnification Med Magnification

Review
H&E Stained
Light Micrograph

High Magnification
Histology Methods [Link]

Some cellular components such as

CARBOHYDRATES are neutral in charge, will
not bind to H&E, and look colorless.

Other components such as

LIPIDS are lost during tissue
preparation, and look colorless.

Special stains and techniques are needed

 Histology Methods [Link]

Special Stains & Techniques

Silver Nitrate
Stains neurons
Periodic Acid-Schiff (PAS
Special magenta colored stain used to
visualize carbohydrates

Osmium-Fixation
Retains lipids during preparation
 Histology Methods [Link]

Overview
Histology Methods
REVIEW OUTLINE

Histology Methods 1. Microscopy

● Light Microscopes
● Transmission Electron Microscopes
● Scanning Electron Microscopes
Review Questions ● Units of Measure
• When would you prefer to use
TEM instead of LM? SEM? 2. Tissue Preparation
• What does a basophilic ● Fixation and Sectioning
cytoplasm indicate about a ● Histochemistry: Four Basic Molecules
cell’s function? ● Staining: H&E
Special Techniques
• What stain would you use to ●

visualize mucus?
REVIEW OUTLINE

Histology Methods 1. Microscopy

● Light Microscopes
Practice Questions ● Transmission Electron Microscopes
● Scanning Electron Microscopes
● Units of Measure

2. Tissue Preparation
● Fixation and Sectioning
● Histochemistry: Four Basic Molecules
● Staining: H&E
● Special Techniques
 Histology Methods [Link]

Knowledge Check
The dye used to stain this image is
best used to visualize:

A. Structures with a net +ve charge

B. Mitochondria and lysosomes
C. Stored protein vesicles
D. Cytoplasmic ribosomes
E. Mucus and glycogen
F. Eosinophilic structures
 Histology Methods [Link]

Knowledge Check
Which of the following staining methods will best highlight changes in
levels of stored glycogen in liver cells under fasting and fed conditions?

A. Hematoxylin and Eosin

B. Periodic Acid-Schiff
C. Silver nitrate
D. Osmium Fixation
E. Metal stain with TEM
REVIEW OUTLINE

1. Plasma Membrane
Overview of Cells:
Organelles 2. Cellular Organelles
● Nucleus
● Nucleolus
● Rough ER
● Smooth ER
● Golgi Apparatus
● Mitochondria

Histology Slides Licensed From © Histology Guide

 Overview of Cells [Link]

Plasma Membrane

Lipid Bilayer
Hydrophilic polar heads
Hydrophobic core of fatty acids

Cell Borders in LM
Visible due to transmembrane
proteins that pick up eosin stain

H&E
Overview of Cells [Link]

Plasma Membrane in EM
Hydrophobic layer stains light
hydrophilic layers stain dark

TEM TEM
 Cellular Organelles [Link]

Nucleus Euchromatin EC Transcriptionally

Nuclear envelope (membrane bound organelle) active DNA, loosely packed.
has pores for communication with cytosol.
Heterochromatin HC Inactive
DNA, tightly packed (dense & dark).

Average diameter: 10 15 μm

TEM H&E
 Cellular Organelles [Link]

Nucleolus
Nucleus contains a nucleolus for formation of ribosomal RNA,
prominent in active secretory cells with lots of euchromatin.
 Cellular Organelles [Link]

Rough Endoplasmic Reticulum (rER)

Composed of stacks of flat cisternae
Has ribosomes attached to the outer membrane surface for protein synthesis
Highly developed in secretory cells, basophilic because of ribosomes
 Cellular Organelles [Link]

Rough ER
Ribosomes on rough ER give rise to basophilic cytoplasm in LM

TEM H&E
 Cellular Organelles [Link]

Smooth Endoplasmic Reticulum (sER)

Tube-shaped cisternae, continuous spaces with flat cisternae of rER
Lacks ribosomes, involved in synthesis of steroid hormones and phospholipids

Nucleus

Smooth ER
Rough ER
[Link]
 Cellular Organelles [Link]

Smooth ER
Smooth ER lacks ribosomes, poorly staining with H&E

TEM
Cellular Organelles [Link]

Golgi Apparatus
Modification of proteins, then
packaged onto transfer vesicles.
Poorly staining with H&E

TEM
 Cellular Organelles [Link]

Mitochondria
Double membrane organelle containing enzyme system that
generates ATP Mitochondria are abundant in cells with high energy
requirement (ex. Ion pumping cells)

Eosinophilic staining in LM (due to enzyme content).

 Cellular Organelles [Link]

Overview
Cell Organelles
 Cellular Organelles [Link]

Practice
Cell Organelles

H&E
 Cellular Organelles [Link]

Knowledge Check
Identify the material indicated by the
purple box, be specific. Describe the
process occurring in this region.
 Cellular Organelles [Link]

Knowledge Check
You receive an H&E stained sample for histopathological study and
notice that it consists of clusters of cells with cytoplasmic regions of
both basophilia and intense eosinophilia. The nuclei are highly
euchromatic and contain prominent nucleoli. Which of the following
most likely describes the function of these cells?

A. Lipid storage
B. Glycogen storage
C. Protein secretion
D. Ion pumping
E. Lipid hormone secretion
REVIEW OUTLINE

1. Plasma Membrane
Overview of Cells:
Organelles 2. Cellular Organelles
● Nucleus
● Nucleolus
● Rough ER
● Smooth ER
● Golgi Apparatus
● Mitochondria
REVIEW OUTLINE

1. Cytoplasmic Inclusions
Overview of Cells: ●
●
Protein Vesicles
Mucus
Cytoplasm ● Glycogen
● Lipids

2. Cytoskeleton
● Microfilaments
● Microtubules
● Intermediate Filaments

Histology Slides Licensed From © Histology Guide

 Cell Cytoplasm [Link]

Protein Vesicles
Secretory Granules: Cells store proteins (or other substances) in membrane-bound
vesicles until signal is received to exocytose substance out of cytoplasm.

TEM H&E
 Cell Cytoplasm [Link]

Protein Vesicles
Secretory Granules: Cells store proteins (or other substances) in membrane-bound
vesicles until signal is received to exocytose substance out of cytoplasm.

Other cytoplasmic vesicles:

Lysosomes or phagosomes
 Cell Cytoplasm [Link]

Mucus
Carbohydrate-rich substance, acts as a lubricant along cell surface. Specialized cells store
mucus in membrane-bound vesicles until signal is received to secrete substance.

H&E PAS
 Cell Cytoplasm [Link]

Cytoplasmic Inclusions
Not enclosed in plasma membrane

Glycogen
Carbohydrate (energy) storage (liver, muscle)

H&E PAS
 Cell Cytoplasm [Link]

Cytoplasmic Inclusions
Not enclosed in plasma membrane

Lipid droplets
Lipid storage in steroid hormone secreting cells or adipocytes
 Cell Cytoplasm [Link]

Cytoskeleton

Three types of protein filaments

Microfilaments
Microtubules
Intermediate Filaments
 Cell Cytoplasm [Link]

Cytoskeleton
Microfilaments
Globular actin proteins assemble to form filaments (7nm)
Highly dynamic (constant remodeling of length)

Function: Cell motility, tracks for the movement of motor

proteins (Myosin), maintains cell shape
Cell Cytoplasm [Link]

Cytoskeleton
Microfilaments
Maintain shape of microvilli (finger like extensions)

TEM TEM
 Cell Cytoplasm [Link]

Cytoskeleton
Microtubules
Globular tubulin proteins assemble into a hollow tube 25nm)
Highly dynamic (constant remodeling of length)

Function: Cell motility, tracks for the movement of motor

proteins (Kinesins and Dynein), maintains cell shape.
 Cell Cytoplasm [Link]

Cytoskeleton
Intermediate Filaments
Multiple strands of fibrous proteins coiled together
into strong rope-like filaments (10 nm)

Function: Provide high tensile strength to cell,

maintain shape and anchor organelles in place.
 Cell Cytoplasm [Link]

Overview
Cell Cytoplasm
 Cell Cytoplasm [Link]

Knowledge Check
What do the cytoplasmic inclusions within these
two different cell types have in common?
 Cell Cytoplasm [Link]

Knowledge Check
Consider a cellular modification where you extend the cytoplasm to
form a finger-like process. To perform its function this extension needs
to be filled with a cytoskeletal fiber that is both dynamic (able to modify
its length) and strong. Which cytoskeletal fiber should it contain?

A. Microfilaments
B. Actin filaments
C. Microtubules
D. Intermediate Filaments
E. Keratin filaments
 Cell Cytoplasm [Link]

Knowledge Check
A 28-year old female suffered a miscarriage at 5-month gestation. She is
seeking genetic counselling to understand the cause of death of her child.
Histopathological studies revealed abnormally shaped cells from various
tissues. EM examination show decreased density of terminal web, abnormal
core of microvilli and fewer cytoskeletal tracks for myosin motor proteins. Which
of the following cellular fibers is most likely affected?

A. Microfilaments
B. Collagen fibers
C. Microtubules
D. Intermediate Filaments
E. Keratin filaments
REVIEW OUTLINE

1. Cytoplasmic Inclusions
Overview of Cells: ●
●
Protein Vesicles
Mucus
Cytoplasm ● Glycogen
● Lipids

2. Cytoskeleton
● Microfilaments
● Microtubules
● Intermediate Filaments