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hu

Introduction to histology.
Microscopic techniques. Basic
tissue types
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Histology
Histology (microscopic anatomy): study of tissues and how they
form organs
• Greek histos „tissue“ and logos „science“
• Whole body contains only 200 different types of cells
• Four basic tissue classes
• Tissue: cells and intercellular substance specialized for a
specific function
• Organ: structure with discrete boundaries
• composed of 2 or more tissue types
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Discovery of microorganisms

• Antony van
Leeuwenhoek
(1632-1723)
– first person to
observe and
describe
micro-
organisms
accurately
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Scale
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https://learn.genetics.utah.edu/content/cells/scale/

Size:
50 cm …………. 5 cm ………. 5 mm ……….. 0,5 mm= 500 μm

50 μm ….. 5 μm…… 0,5 μm=500 nm light microscope

50 nm …………. 5 nm ……….. 0,5 nm electron microscope

„million‐volt field emission transmission electron
microscope (FE‐TEM)”: 0,05 nm

Use the measures and units properly!

1 μl = 1 mm3, 1 ml = 1 cm3,
1 μm= 10 ‐6 m
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• The thickness of our slides is between 5 and 10 μm (sometimes 50‐100 μm)

• The electron microscopic slides are 0,01–0,5 μm thick, they are rarely used in
the everyday practice.
• Most of our slides are dyed, but occasionally we examine unstained
preparations.
• What is the origin of the tissues examined?
– we examine well preserved, „healthy” tissues
– cadavers
– rarely from animals
– human tissues obtained during operations (eg. stomach, gut, kidney)

• Pathologists have „no choice”:

– excision – eg. naevus, mole, cysts
– biopsy – breast tissue, stomach, gut, liver, kidney, etc.
– tissues obtained during pathologic dissection (eg. tissue from myocardial
infarction, tumors, etc.
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Objectives
To understand:
– How cells and tissues are arranged in the normal organ system of the
body, and
– How these cells and tissues are specialized to perform the function(s)
most effectively.

The knowledge gained will hopefully provide a cellular and ultrastructural

“framework” for all of the other topics (anatomy, embrology, physiology,
biochemistry, etc.) that you’ll learn soon.

Histology is also, of course, a FUNDAMENTAL part of PATHOLOGY.

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Tissues
• Cells work together in functionally related
groups called tissues
• Types of tissues:
1. Epithelial tissue
2. Connective and supportive tissue
3. Muscular tissue
4. Nervous tissue
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Correlate
Structure
and
Function
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Differences among tissue classes

1. Types and functions of cells
2. Characteristics of the matrix (extracellular
material)
• Rubbery, stony, or gelatinous
3. Relative amount of space occupied by cells
versus matrix
• CT vs. muscle and epithelium
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Tissue Sectioning
1. Preparation of histological specimens
– fixation
– sections
– mounted on slides & stained

2. Sectioning (slicing) an organ or tissue reduces

a 3-dimensional structure to a 2-dimensional
slice
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What is microscope?
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What is microscope?
Theoretically a microscope is an array of two lenses.

Focal plane Image plane

Image plane

Objective Tube lens Eyepiece

lens lens

Modern microscope with ICS

(Infinity Colour corrected System)

Classic compound microscope

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Light microscopy

1. ILLUMINATION SOURCE
2. CONDENSER LENS
3. SPECIMEN STAGE
4. OBJECTIVE LENS
5. PROJECTION (OCULAR) LENS
6. OBSERVER

• CELLULAR FEATURES ARE STAINED

DIFFERENTIALLY BASED PRIMARILY UPON
CHEMICAL PROPERTIES.
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Tissue Preparation
for light microscopy
1. Stabilize cellular structures by chemical fixation.
2. Dehydrate and infiltrate tissues with paraffin or plastic.
3. Embed fixed tissues in paraffin or plastic blocks.
4. Cut into thin slices of 3-10 micrometer thick; collect sections
on slides.
5. Re-hydrate and stain with Hematoxylin (a basic dye): Stains
basophilic structures (e.g. nucleic acids) blue/purple.
6. Counter-stain with Eosin (an acidic dye): Stains acidophilic or
“eosinophilic” structures (e.g. proteins, membranes)
red/pink.

“H & E” staining is routine, but other dyes and staining

techniques may be used to visualize other structures.
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Tissue Preparation
for light microscopy
• Tissue sampling (1 cm3).
• Fixation (most often 10 %-os neutral or buffered formaldehyde)
• Removal of fixative with water or buffer
• Dehydration in ascending alcohol (water phase- 30%-50%-70%-96%-absolute alcohol)
• Embedding
– During this step the material is prepared for cutting sections from it with a microtome. Most often we
use celloidine-paraffin embedding, which is time consuming but gives a good structure.
– Other possibilities would be e.g. paraffin embedding, or to make the slides without embedding (frozen
sections from deep-frozen tissues or vibrotome sections on room temperature. Each process has its
advantages, we use them according to our needs, depending what and how fast we would like to stain.
(E.g. in pathology sometimes very quick methods are needed that give result in extremely short time
during an operation.)
• Cutting
– The sections are made manually, with microtome (temnein/Greek/=cut).
– In our case the embedded material is mounted on a wooden cube (specimen holder)and fixed into the
microtome. The sections are made with a very sharp knife. The mechanics of the microtome lifts the
material with the adjusted thickness (5-10 micrometer usually) after each cut. The sections are
mounted on glass slides.
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Tissue Preparation
for light microscopy
• Staining
– First the embedding material has to be removed with organic solvents.
– The slides are rehydrated in descending alcohol.
– We stain the slide, most often with haematoxyline-eosin (HE).
• Haematoxyline is a basic dye, stains the acidic structures blue (DNA, RNA - nucleus, RER, ribosomes). Things that
stain with haematoxyline are basophylic. Eosin is an acidic dye, basic components stain with it in pink-red color
(cytoplasm many times, connective tissue fibers): this is the acidophylic or eosinophylic staining.

– To increase contrast and specificity an often used step is the differentiation: the loosely and not
specifically adhering dye molecules are washed off (e.g. in the case of haematoxyline with HCl-alcohol
mixture).
– We might use other dyes too, like AZAN, iron-haematoxylin, Scharlach, PAS, orcein, OsO4, etc. Melanin
containing epithelial- and connective tissue cells are shown in unstained preparations.
• Dehydration of the sections in ascending alcohol.
• Xylol treatment makes the slides more transparent
• Mounting
– This means that for protections the sections have to be covered with a thin glass plate (cover glass). This
cover-glass has to be glued on with a special material the refractive index of which is the same as that of
the glass. Previously we used a natural material (Canada-balsam) today synthetic compounds are used
(e.g. Permount).
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Light microscopy
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Transmission electron microscopy

1. ILLUMINATION SOURCE (generates
electron beam)
2. CONDENSER LENS
3. SPECIMEN STAGE
4. OBJECTIVE LENS
5. PROJECTION LENS
6. FLUORESCENT VIEW SCREEN
7. VIEWING WINDOW & OBSERVER
• YIELDS A 2-DIMENSIONAL IMAGE CAPABLE
OF 0.2 nm RESOLUTION.
• CELLULAR FEATURES ARE STAINED WITH
ELECTRON-DENSE, HEAVY METAL STAINS
YIELDING ONLY A BLACK AND WHITE IMAGE
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The challenge:

3D structures, but
viewed only in 2D…
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1 2 3 4 5
•Slices 1 & 5
miss the yolk
1 5 / cell nucleus

•Cell nucleus
2 3 4
is smaller in
sections 2 &
4
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A B • Image A is a cross
section of elbow
macaroni, resembling
a blood vessel, piece
of gut, or other
tubular organ.
• Image B is a
longitudinal section
of a sweat gland.
Notice what a single
slice could look like
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Types of tissue sections

• Longitudinal section
– tissue cut along the
longest direction of an
organ
• Cross section
– tissue cut perpendicular
to the length of an organ
• Oblique section
– tissue cut at an angle
between a cross &
longitudinal section
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Microscopy
Virtual Microscopy

Microscope Slides
Digital Images
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Virtual Microscopy
• Glass microscope slides line-scanned
using a computer-controlled
microscope
• Line scans compiled into single “digital
slide” that may be 200k x 200k pixels
(that’s 40 GIGApixels!)
• Digital slides stored as compressed files
(~1,5 GB) and delivered via Web or file-
server
• Digital slides viewable as flash objects
within web browser or in proprietary
format
• Any region of interest on digital slide
may be viewed at a range of
magnifications with resolution up to
0,25μm/pixel
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Histology Guide
http://histologyguide.com/slidebox/slidebox.html
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UPMS histo slides

https://an-slides.aok.pte.hu/en

Login: anat2018
Password: tomy2018
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Microscopes and glass slides

still have their place!
• The focal plane and depth-of-field (aperture) of the digital
slide is fixed
• The digital slide is only a representative specimen
• Servers crash
• Knowing how to use a microscope has its value!
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Microscope Resolution
• ability of a lens to separate or distinguish
small objects that are close together
• wavelength of light used is major factor in
resolution
shorter wave-length  greater resolution
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• working distance
— distance between the front surface of lens and surface of cover glass or
specimen
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Refraction
• Change in the direction
of a wave (light) due to
a change in speed
• The straw in the picture
looks bent because the
light is bending as it
moves from the water to
the air
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Refractive Index (RI)

• RI of a material a measure of
the speed of light in material

• RI is the ratio of the velocity

of light in a vacuum to the
speed of light in the specified
material

• Used in calculating focusing

power of lenses and dispersion
properties of prisms
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 Always carry a microscope with one hand
holding the arm and one hand under the base. an-server.pote.hu
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Ocular lens
(Eyepiece)
Body Tube

Nosepiece
Arm
Objectives

Stage
Stage Clips
Coarse Adjustment
Diaphragm
Fine Adjustment
Light

Base
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Comparing powers of magnification

We can see better details with higher the

powers of magnification, but we cannot see
as much of the image.
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What’s my power?
To calculate the power of magnification, multiply the power of the ocular lens by the power
of the objective
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