Biological preparations for TEM:

Ultra-thin cut:

In transmission electron microscopy, the sample must first be:

cut into ultra-thin slices 20-100 nm. thick, and impregnated
with heavy metal salts that will: bind differentially to different
intracellular structures make them more or less opaque to
electrons.

Metallic shading

The sample is coated with a thin layer of heavy metals. thus

The cellular structures appear more or less dense (black and
white) on a light.

Background cryodeblasting

Frozen samples are fractured along the lines of greatest

weakness (i.e., the middle of the inner membranes).
The cryodeblasting technique
Negative staining

In transmission electron microscopy: the object can only be

visible if, after fixation, it is impregnated with substances

containing atoms of heavy metal opaque to electron.

Fixation and subsequent reactions can modify significantly

Nucleocapsid virus measles

the structure of the biological object.

The negative contrast makes it possible to darken the

background without coloring the object itself and therefore

to make it visible without modifying it, by simple contrast.

The substance opaque to electron is deposited around the Human Papillomavirus

- Biological preparations for SEM
- Biological material: cells, bacteria and viruses.
-These structures are fixed, dehydrated and covered with a thin layer of metal (Or,
palladium, platinum) vaporized under vacuum.
- The metallized surface of the sample is regularly scanned by an electron beam.
There, the raised surfaces lead to the emission of secondary electrons (bright spots)
and the depressions are without emission of electrons (appears in dark points)
- The emitted electrons are collected by an electron detector then converted into an
image by an electron converter. The final image appears in 3 dimensions on a
television screen.
Cell membrane
 Cell membranes
•Boundary between the inside and the outside of the cell.

•They allow cells to maintain an internal environment

different from the extracellular environment.

•They have a very selective permeability.

•The membrane surface inside the cell is often larger than

the surface around the cell.

•The main constituents are lipids and proteins.

•Area of plasma membrane 700 µm2

Schema of a cell and its membranes:
•Area of all internal membranes 7000 µm2 2. Nuclear membrane; 4. Vesicle;

•Thickness: 7 to 8 5. Rough endoplasmic reticulum;

nm. (1 nm = 1/1000 6. Golgi apparatus;

8. Smooth endoplasmic reticulum;
de µm)
9. Mitochondria;
•Two sheets visible 10. Vacuole; 11. Cytoplasm;
under an electron 12. Lysosome
microscope.
Plasma membrane functions
Compartmentalisation (separation of the outside and the inside of the cell).

Exchanges of information with other cells (hormone receptors, gap junctions).

Regulation of the transport of ions, proteins, sugars, fats, etc.

Cell movements (pseudopodia, endocytosis-exocytosis).

Recognition phenomena (surface antigen).

Regulation of metabolism (intracellular transduction of extracellular signals).

Provides a site for chemical reactions that cannot occur in an aqueous

environment .

Plasma Membrane Compositions:

Lipids: (Phospholipids and cholesterol) (~49%): Form the skeleton

of the membranes.

Proteins: (receptors, transporters, enzymes)(~ 45.%): Attached more or less to

phospholipids.

Carbohydrates: (glycophospholipids and glycoproteins) (~1.6-8%).

 Fluid mosaic model
(model of "Singer and Nicholson" (1970))
•Two layers of phospholipids.

•Proteins on the surface and through.

•Polysaccharides (carbohydrate) attached to lipids or proteins.

•Cholesterol between phospholipids

Structure of the membrane:
Lipids are organized in a double layer thanks to their ambiphilic
properties. In some cases, certain lipids organize themselves into
vesicles called liposomes and micelles when they are in an aqueous
medium. Lipids move in the plane of the membrane, but a protein
cannot change orientation.
 I/ Membrane lipids
1- Hydropilic pole (polar): forms a flexible film (surface) composed by a phosphate
plane esterified by a polar molecule (ethanolamine, choline, serine) and connected to
a hydrophilic molecule such as glycerol.
2- Hydrophobic pole (apolar): Film of fatty acids (FA) which can be saturated or
unsaturated. (guarantees to the membrane a coherence between resistance and
flexibility. On the external and internal faces (hydrophilic poles) are fixed extrinsic
proteins on which the cytoskeleton clings (on the internal side) or the molecules of the
extracellular matrix (external side).
 Structure of lipides (GPL)

G
FATTY ACID
L
FATTY ACID
Y

C
ALCOHOL PHOSPHATE
E

L
 glycerol

H2C OH

HC OH

HO CH2

glycerol
 two fatty acid

O palmitate

H2C O C (CH2)14 CH3

HC O (CH
C 2)7 C C
(CH2)7
CH3
O H H
HO
CH2 oleate

glycerol Fatty acid

 one phosphate

O palmitate

H2C O C (CH2)14 CH3

HC O C
(CH ) (CH2)7 CH3
2 7 C C
O
H H
O
HO P O
CH2 oleate

O-

phosphate glycerol Fatty acide

 One alcohol

O palmitate

H2C O C (CH2)14 CH3

HC O C
(CH ) (CH2)7 CH3
2 7 C C
H3C O
H H
O
H3C CH2 CH2 O P
N+ oleate
O CH2
H3C
O-

alcohol phosphate glycerol Fatty acid

 Fatty acids are the key constituents of lipids

Fatty acids are carboxylic acids characterized by a repetition of methylene

groups -CH2- forming a carbon chain

Aliphatic (carbon) chain Acid

Natural fatty acids have an even number of carbons: C14->C24

They can be saturated or unsaturated.
Saturated fatty acids are linear.
Unsaturated fatty acids create a bend in the structure.
 Example of saturated fatty acid nomenclature

•The common name recalls the origin.

The systematic name recalls the structure (nb carbons, of establishment).
•From C10 they are insoluble in water and solid at ambient temperature.
Example of the nomenclature of unsaturated fatty acids

1 2
CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)7-COOH
3
9 …. 1

α linolenic acid: abbreviation C18 :3 ε 6 9 or C18: 3 ∆ 9,12,15

Translate 18 carbon atoms (C18)

3 double bonds (:3)

 …Quiz of nomenclature

C18 : 1 ω 9 ou C18 : 1 ∆
9

Oleic acid

linolenic-α-acid C18 : 3 ω 3,6,9 ou C18 : 3

∆ 9,12,15
C20 : 4 ω 6,8,10,12 ou
C20 : 4 ∆ 5,7,9,11

Arachidonic acid
Impact on the cohesion between chains
The presence of a double bond is important for the macroscopic behavior of fatty
acids. The double bonds contribute to weaken the interactions between the close
chains thus the membrane becomes more fluid (flexible)

Cohesion of membrane phospholipids

The cohesion of membrane phospholipids is due to Van Der Waals bonds between fatty
acids and hydrophobic interactions between fatty acids
Membrane fluidity:
Lipid mobility is necessary for cellular activity. They can move in different ways within
the membrane: rotation, lateral diffusion and flip flop (moving from one sheet to
another).
Membrane fluidity is involved in various cellular functions: absorption, secretion,
protection, adhesion, communication, interaction with the matrix, etc.
Fluidity is influenced by various factors:
-The fatty acid composition: The shorter and unsaturated the fatty acid carbon chains,
the more fluid the membrane.
-The proportion of cholesterol: Cholesterol reinforces the strength and rigidity of the
membrane and corresponds to up to 50% of the total lipids of the membrane.
-The number of proteins: Proteins decrease membrane fluidity. Some proteins will be
blocked by intracellular or extracellular structures (interactions: protein/protein or
protein/cytoskeleton).
.-Temperature: an increase in temperature causes the fluidification of the membrane
.-The nature of the polar heads (nature of the amino acids).
 Biological membranes contain 4 classes of lipids

1. Glycerophospholipids
2. Sphingolipids
3. Glycolipid
4. Steroid
 1. Glycerophospholipides (GPL)

Glycerophospholipids are named after the alcoholic groups: phosphatidylcholine

 The diversity of glycerophospholipids

NH + OH
H3N+ CH2 OH 3
CH2 OH
ethanolamine OOC
-
C CH2 OH H OH
H
H
serine H
H3C OH HO OH H

OH OH CH2 C CH2 H
H OH
H3C N+
CH2 CH2 OH inositol
glycerol
HC
3 choline

Phosphatidylethanolamine (PE) for ethanoamine

Phosphatidylserine (PS) for serine
Phosphatidylcholine (PC) for choline
Phosphatidylinositol (PI) for inositol
Phosphatidylglycerol (PG) for glycerol
 Phosphatidyl

ethanolamine serin choline Sphingomyélin

e e
CH3 CH3
NH3 NH3 + +CH3 -N- +CH3 -N-
+ H-C-COO
CH3 CH3
CH2 - CH2
CH2
CH2
CH2
O O O
CH
O=P-O O
CH
O=P-O - O=P-O
- - 2
O=P- 2
-
O O
O O O
CH2 - CH - CH2 CH2 - CH - CH2 CH2 - CH - CH2 CH2 - CH - CH2
O O O O O O CH
NH
C=O C=O CH C=O ;
C=O
C=O C=O C=O
1. Glycerophospholipids
2. Sphingolipids
2.1. Sphingophospholipids
2.2. Sphingoglycolipids
3. Glycolipids
4. Steroids
 2. sphingolipides

Sphingolipids constitute a second class of membrane lipids. They are synthesized on

the luminal surface of the Golgi cisterns. Sphingolipids are found next to
glycerophospholipids. The basic constituent is sphingosine, it is an amino dialcohol +
a hydrocarbon chain (CH2)12

sphingosine
 2. Les sphingolipides (SGL)

They differ from GLPs by the presence of a sphingosine instead of glycerol. SGLs
consist of a sphingosine linked to an FA by an amide bond forming a ceramide + a
polar head. We distinguish:

2.1. Sphingophospholipids: the best known is sphingomyelin whose polar head is

made up of choline+phosphate.

2.2. Sphingoglycolipids (without phosphate group).Simple with one or more sugar

residues (glycosyl) in their polar region: Cerebrosides.

Complexes contain several sulphated sugar groups: the Sulfatides.

Complexes contain several sugar groups + Sialic Acid: the Gangliosides.

 Gal GalNac

CH3
+
CH3 -N-
CH3 CH2
Ac Sial Gal
CH2

O
O=P-O
- Gal Glc

O
CH2 - CH - CH2
OH OH
CH
O O
NH
CH - CH - CH2 CH - CH - CH2
CH C=O
CH2 CH2 C=O
NH NH
CH2 CH2
C=O

Sphingomyelin is important Single

for cell signal transmission Ganglioside
galactocerebroside GM1 SGL
and intercellular recognition
SGL complex
Cholera toxin
receptor
1. Phospholipids
2. Sphingolipids
3. Glycolipids : 2FA + glycerol + sugar
4. Steroids
 3. Glycolipids (relatively in small quantity)
Diacylglycoglyceride

Similar to phospholipids (GLPs) in composition except that they are:

- Phosphate free

- Their glycerol is esterified by 2 FA on the alcohol functions 1 and 2 of glycerol.

- On the alcohol function at position 3 of glycerol there is a sugar which constitutes

the polar head (galactose, etc.);

- Simple GLPs with a single sugar residue (glycosyl) in their polar region, exp
diacylglycoglyceride.

- Complex GLPs contain multiple sugar groups.

1. Phospholipides
2. Sphingolipides
3. Glycolipides
4. Stéroïdes
 4.Les stéroïdes
Cholesterol is the main steroid constituent of normal tissues it constitutes about 25%
of membrane lipids

cholesterol ergosterol
The alcohol function
constitutes the polar head animal cells plant cells

Role of membrane cholesterol

- By inserting itself between the molecules of
phospholipids, it stabilizes the membranes by
avoiding excessive fluidity.
- Synthesis in the liver or nutrient supply and
transport in the blood.
- Cholesterol is the precursor of steroid
hormones.
 Physical structure of the fluid membrane bilayer

1. Fluidity of membranes: Lipids are mobile within bilayers

Lateral displacement
of lipids (+fast)
V=2µm/s
Factors Affecting Membrane Fluidity Consequence of this fluidity
1- Temperature Can repair itself
2- The length of the hydrocarbon chains Can vary its size easily
3- The number of double bonds Allows a sphere to change
4- The cholesterol content which
stiffens the bilayer
🡩 cholesterol🡪 fluidity↓ at T° amb
5- The number of proteins:.
2. The distribution of lipids is asymmetric within the same
membrane
OUTER layer: more saturated fatty acids
phosphatidyl-choline; Glycosphingolipides
INTERNAL layer: highly unsaturated fatty acids
phosphatidyl-serine; phosphatidyl-ethanolamine
Exemple: plasmic membrane

outside inside
Phosphatidylserine 0 100
Phosphatidylethanolamine 10 90
Phosphatidylcholine 90 10
Glycolipides 100 0
Cholestérol 75 25
 3. The lipid composition of the membranes

Membrane Mitochondrie Réticulum E.Coli

proportion en plasmique (memb. Externe) endoplasmique
%
Phosphatidylethanolamine 16 23 16 80
Phosphatidylserine 6 2 3 Trace
0
Phosphatidylcholine 17 50 55 Trace
Phosphatidylinositol <1 0 0 Trace
Sphingomyeline 17 5 3 Trace
0
Glycolipides 2 0 0
Cholestérol 45 <5 6

Glycolipids only in plasma membranes of eukaryotes