Transmission Electron Microscopy

Transmission Electron
Microscopy
Presented by:
Maha Hammady
MBBCh, Demonstrator at Medical Histology & Cell
Biology Department,
Faculty of Medicine Alexandria university
Maha Hammady 4-1-2021

Transmission electron microscopy (TEM) is a significant tool in demonstrating
the ultrastructure of cells and tissues both in normal and disease states. In
particular, TEM can be crucial in the diagnosis of various renal pathologies,
the recognition of subcellular structural defects or the deposition of
extracellular material

Tissue preparation for transmission
electron microscopy
-to obtain a high-quality image
and optimize the resolution of
the instrument, it is necessary to
section the tissue to a thickness
of around 80 nm.
-Sectioning at this level requires
tissues to be embedded in a rigid
material which can withstand
both the vacuum in the
microscope column and the heat
generated as the electron beam
passes through the section. The
most suitable embedding
material is the resins

1-Specimen handling
-it is crucial that samples are fixed as soon as possible after the biopsy is
taken.
-The standard approach is to immerse the specimen in fixative immediately
on collection.
-Once in fixative, the specimen is cut into smaller samples using a scalpel or
razor blade. The final tissue blocks are in the form of small cubes
(approximately 1 mm3).

1-Specimen handling
-Manual tissue processing is best performed by keeping the tissue sample in
the same vial throughout, and using a fine pipette to change solutions.

2-Fixation
-The fixatives used in TEM generally comprise a fixing agent in buffer (to
maintain pH). The standard protocol involves primary fixation with an
aldehyde, usually glutaraldehyde, to stabilize proteins, followed by secondary
fixation in osmium tetroxide to retain lipids this is termed (double fixation).
-4F1G Fixative is the most commonly used primary fixative, it is formed of (4%
Formaldehyde & 1% Glutaraldehyde phosphate buffered saline, pH 7.4). using
this aldehyde mixture overcome the disadvantages of glutaraldehyde (a slow
penetration rate) and formaldehyde (less stable fixation) when applied
individually.
-The use of cold primary fixative (4°C) helps to minimize postmortem
changes.

2-Fixation
Crosslinking is the process of chemically joining two or more molecules by a covalent
bond. ... Attachment between groups on two different proteins results in
intermolecular crosslinks that stabilize a protein-protein interaction.

2-Fixation
-Volume of fixative should be at least 10 times
the volume of the tissue and it is also vital to
ensure that the tissue remains completely
submerged in the fixative.
-The time required for optimal fixation depends
on a range of factors. These include the type of
tissue, the size of the sample. In most
circumstances immersion of 0.5–1.0 mm3 blocks
of tissue for 2–6 hours is sufficient.
-Specimens fixed in aldehyde solutions should be
washed thoroughly in buffer before post-fixation
in osmium tetroxide to prevent interaction
between the fixatives which can cause
precipitation of reduced osmium.

-The use of osmium tetroxide fixation to preserve
lipid is fundamental to TEM .it is usually used as a
secondary fixative at a concentration of 1 or 2%,
the penetration rate of osmium tetroxide is also
higher in stabilized tissue. So, immersion for 60–90
minutes is sufficient for most specimens. Osmium
tetroxide is generally used at room temperature.
-Osmium tetroxide is usually supplied in crystalline
form, sealed in glass ampoules. Extreme care
should be exercised when preparing this material,
gloves and eye protection should always be worn. It
is essential to only handle osmium tetroxide in a
fume-hood, as the vapor will also fix other tissues,
including the eyes and nasal tissues of the handler.
2-Fixation

-The most sensitive cellular indicators of autolytic/degenerative change are
mitochondria and endoplasmic reticulum, both of which may show signs of
swelling (a reflection of osmotic imbalance) only a few minutes after the cells
are separated from a blood supply
2-Fixation

3-Dehydration
-epoxy resins are immiscible with water and specimens must be dehydrated
prior to resin infiltration.
-Dehydration is performed by passing the specimen through increasing
concentration of an organic solvent to prevent the damage which would
occur with extreme changes in solvent concentration.
-The most frequently used dehydrants are acetone.

4-Embedding
-Embedding mixture is prepared by mixing of Epoxy resin (araldite),
plasticizer, hardener and accelerator in a flask. The mixture is kept in oven at
60 oC for 10 minutes to eliminate air bubbles.

4-Embedding
-After dehydration the tissue is infiltrated
with liquid resin mixture. The resin is
introduced gradually, beginning with a
50:50 mix of transition solvent (propylene
oxide) and resin followed by a 25:75 mix,
then finally pure resin.
- An hour in each of the preliminary
infiltration steps is usually adequate,
although some recommend leaving
samples in pure resin for 24 hours.
-Gentle agitation using a low-speed,
angled rotator during these steps will assist
resin infiltration.

-Once infiltrated, tissue samples are placed in an appropriate capsule or mold
(various shapes and sizes are available) which is filled with resin. The resin is
polymerized using heat. Soft polyethylene capsules (resistant to 75°C) are
recommended for general embedding.
4-Embedding
Embedding mold Embedding capsule

During polymerization, epoxy resins form cross-links, creating a three-
dimensional polymer of great mechanical strength. As well as their properties
of uniform polymerization and low shrinkage (usually less than 2%), epoxy
resins also preserve tissue ultrastructure, are stable in the electron beam,
section easily and are readily available.
4-Embedding

5- Trimming
Once polymerized, blocks must be cleared of excess resin (trimmed) to
expose the tissue for sectioning. The final trimmed area should resemble a
flat-topped pyramid with a square or trapezium-shaped face

6- Sectioning
Ultra-microtomes diamond knives are used to get an ultra-thin section
(approximately 60 nm). thin sections are floated out for collection as they are
cut

7- Mounting
-The ultra-thin sections are mounted on copper grids (200 square mesh) 3.5
mm in diameter

8-Staining
-Grids are placed on filter-paper in petri-dishes and allowed to dry.
- After drying, sections are stained with urinyl acetate for 10 minutes, and
then they are rinsed with distilled water before being stained by lead citrate
for another 10 minutes.
-This staining step increases contrast pf the resulting images

9- Examination and photography
-Sections are examined and photographed by TEM

The Mitochondria

• Mitochondria are the cellular organelles
responsible for generating energy derived
from the breakdown of lipids and
carbohydrates, which is converted to ATP by
the process of oxidative phosphorylation.
• Mitochondria were among the first subcellular
organelles examined by electron microscopy.
In the early 1950s
The Mitochondria

Ultra-structure of mitochondria:
• Mitochondria are surrounded by a double-
membrane system, consisting of inner and outer
mitochondrial membranes separated by an
intermembrane space .The inner membrane
forms numerous folds (cristae), which extend into
the interior (or matrix) of the organelle. Each of
these components plays a distinct functional role,
with the matrix and inner membrane
representing the major working compartments of
mitochondria

Ultra-structure of mitochondria:

Shape and organization of cristae
On the basis of their appearance in may be
broadly classified as either lamellar, tubular:
1-The lamellar form is the commonest ,cristae
have are parallel to one another, and frequently
they are orientated perpendicular to the long
axis of the mitochondrion.
2-Tubular cristae may be circular in cross-
section and they have uniform internal diameter

Drawings to compare the appearance of sections through lamelliform
and tubular cristae.

Electron micrograph of section of rat brown adipocyte tissue,
showing the regular arrangement of the cristae the majority of
which extend the full width of the mitochondrion

Electron micrograph of section of human uterine mucosa epithelial cell in
the middle of the menstrual cycle, showing mitochondrion with parallel
arrays of cristae characteristically in two groups, the intervening matrix
contains DNA.

Concentrically arranged cristae have been observed in cardiac
muscle in invertebrate ,mammalian spermatids ,fascicular zone
of the mouse adrenals and some skeletal muscles .In some of the
latter the cristae membranes show a wavy outline with abrupt,
sharp angles.
Electron micrographs of
mitochondria in the
peripheral cytoplasm of
red fibers in rat soft
palate muscle.

Tubular cristae (arrowheads) are present in many cell types, being a common form in
steroid-producing cells. Monkey Leydig cell

• The prismatic tubular cristae in the hamster
brain mitochondria form a hexagonal lattice
Electron micrograph of mitochondrion in a
section of an astrocyte from hamster
brain.

• tubulovesicular

Distribution:
• Ordinarily mitochondria are evenly distributed
in the cytoplasm. They may, however, be
localized in certain regions

Distribution:
1-proximal convoluted tubules:
they are found in the basal region.

Distribution:
2-distal convoluted tubules:
they are found
in the basal region.

Distribution:
3-In cardiac muscle:
1-Perinuclear mitochondria are present at nuclear
poles, they are spherical.
2-interfibrillar mitochondria are elongated and they
present between the myofibrils forming
longitudinal rows. IFM occupy the entire space
between Z-lines.
3-Subsarcolemmal mitochondria present beneath
sarcolemma, they are variable in their shapes, they
can show oval, spherical, polygonal, or horse-shoe
patterns.

Distribution:
3-In cardiac muscle:

Distribution:
4-In skeletal muscles :

Distribution:
4-In skeletal muscles :
Electron microscopy imaging of
muscle fibers from a patient with
mitochondrial myopathy. from a
patient carrying a deletion
associated with Kearns-Sayre
syndrome. Note the dark, granular
structures indicated with the white
arrows. These inclusions are
aggregates of diseased
mitochondria and are a
characteristic signature of
mitochondrial dysfunction .

Distribution:
5-In sperms :
the mitochondria lie in the middle piece of the
sperm forming the mitochondrial sheath.

Distribution:
6-in close association with lipid droplet:
One of the frequently observed associations is
between mitochondria and lipid droplets
Rat hepatocyte mitochondria, showing close
association with a lipid droplet

Distribution:
7-proximity to sites of
energy utilization:
in ciliated cells for energy
needed for ciliary motion
Human bronchial mucosa, showing
mitochondrial aggregates in the
apical cytoplasm adjacent to the
ciliary border.
X8000

Shape:
• The shape is variable but is characteristic for a
cell or tissue type
• this too is dependable upon environment or
physiological conditions

Size:
• The size of mitochondria also varies. In majority of the cells,
width is relatively constant, about 0.5µ, but the length varies
depends on the functional stage of the cell.
• Megamitochondria: Regardless o f internal configuration,
mitochondria rarely are larger than 1 µm
• in diameter or length. been reported to occur in human
tumors. In the pleomorphic adenoma of the submandibular
gland

Size:
• Megamitochondria: Regardless o f internal configuration, mitochondria
rarely are larger than 1 µ m in diameter or length. been reported to occur
in human tumors. Some o f these megamitochondria contained expanded
cristae.
pleomorphic adenoma of the human submandibular gland: A huge mitochondrion with
arabesque cristae next to the nucleus. Bar :2 µ m

Number:
• The mitochondria content of a cell is difficult to determine, but in
general, it varies with the cell type and functional stage. Thus cells
with high metabolic activity have a high number of mitochondria,
while those with low metabolic activity have a lower number.
• It is estimated that in liver mitochondria constitute 30 to 35 % of
the total content of the cell (normal liver cell contains about 100 to
1600 mitochondria)
• in kidney, 20 % of the total content of the cell
• In lymphoid tissue the value is much lower.
• but this number diminishes during regeneration and also in
cancerous tissue. This last observation may be related to decreased
oxidation that accompanies the increase to anaerobic glycolysis in
cancer.

summary
• Mitochondria are the cellular organelles
responsible for generating energy
• Mitochondria are surrounded by a double-
membrane system, consisting of inner and outer
mitochondrial membranes separated by an
intermembrane space .The inner membrane
forms numerous folds (cristae), which extend into
the interior (or matrix) of the organelle.
• cristae are classified as either lamellar, tubular.
• Mitochondria in general vary in their shape, size,
number and distribution among cells.

Transmission Electron Microscopy

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