Samenvatting EM hoorcollege 3
The electron microscope is a hostile environment for biological samples. Within the EM
electrons are accelerated in high vacuum and have high energy damaging for biological
samples.
Which biological samples can be imaged with TEM:
Tissues
Whole cells
Viruses
Filaments
2D or 3D crystals
Purified macromolecules
In-vivo macromolecules
Sample preperation
The samples for EM are places on so called grids. These consist of two parts:
1. Metal Mesh support and rigidity.
Different material: Copper (toxic for cells) and gold most common
Different mesh sizes and shapes: when the
mesh is to high, the grid is to dense and you
cannot see the sample. When your mesh is
to low, your support film breaks.
You can use a special mesh called a finder mesh. In this mesh are number with which
you can correlate EM and LM images.
2. Support film holds the sample. The most common film is made of amorphous carbon.
(We don’t want crystalline carbon). The carbon support films are electron transparent but
very fragile and hydrophobic (needs pre-treatment).
There are different types of support films:
Continuous used for classical EM, e.g. negative stain
Holey used for cryoEM (freezes faster and less background). Two types
Holy lacey: random distribution holes
Holey circular, square etc.: known shapes of holes
, You also have gold support films. These reduce radiation induces motion but are not
electron transparent.
Your carbon film is made out of amorphous carbon and this is hydrophobic. Your sample is
hydrophilic, so when you prepare your sample you first need to make your support film
hydrophilic. You can do this with Glow discharge – Plasma cleaning. This has 3 functions:
1. Cleaning grids
2. Hydrophilization of the support films
3. Sputtering (only SEM)
Plasma is one of the four fundamental states of matter. It is a gas of ions generated by
energizing gas e.g. heating. Glow discharge is a plasma formed by the passage of electric
current through a gas. It is often created by applying a voltage between two electrodes in a
glass tube containing a low-pressure gas. When the voltage exceeds a value called the striking
voltage, the gas ionization becomes self-sustaining, and the tube glows with a colored light.
The color depends on the gas used (commonly you use air).
When you are doing SEM, you always coat your sample.
The plasma can cause Sputtering: atoms ejected from
solid material. Generated by bombarding the surface with
energetic particles of a plasma or gas. Can be used for
depositing a thin layer (used for SEM sample
preperation).
Sample preperation considerations
Sample protection within the TEM electrons are accelerated in high vacuum and have
high energy damaging for biological samples.
Sample thickness The sample needs to be thin enough for electrons to go through (< 1
um). The penetration depth of electrons increases with increasing acceleration voltage.
Sample contrast biological samples mainly consist of light elements (H, C, N and O)
which weakly scatter electrons and hence show poor contrast.
The electron microscope is a hostile environment for biological samples. Within the EM
electrons are accelerated in high vacuum and have high energy damaging for biological
samples.
Which biological samples can be imaged with TEM:
Tissues
Whole cells
Viruses
Filaments
2D or 3D crystals
Purified macromolecules
In-vivo macromolecules
Sample preperation
The samples for EM are places on so called grids. These consist of two parts:
1. Metal Mesh support and rigidity.
Different material: Copper (toxic for cells) and gold most common
Different mesh sizes and shapes: when the
mesh is to high, the grid is to dense and you
cannot see the sample. When your mesh is
to low, your support film breaks.
You can use a special mesh called a finder mesh. In this mesh are number with which
you can correlate EM and LM images.
2. Support film holds the sample. The most common film is made of amorphous carbon.
(We don’t want crystalline carbon). The carbon support films are electron transparent but
very fragile and hydrophobic (needs pre-treatment).
There are different types of support films:
Continuous used for classical EM, e.g. negative stain
Holey used for cryoEM (freezes faster and less background). Two types
Holy lacey: random distribution holes
Holey circular, square etc.: known shapes of holes
, You also have gold support films. These reduce radiation induces motion but are not
electron transparent.
Your carbon film is made out of amorphous carbon and this is hydrophobic. Your sample is
hydrophilic, so when you prepare your sample you first need to make your support film
hydrophilic. You can do this with Glow discharge – Plasma cleaning. This has 3 functions:
1. Cleaning grids
2. Hydrophilization of the support films
3. Sputtering (only SEM)
Plasma is one of the four fundamental states of matter. It is a gas of ions generated by
energizing gas e.g. heating. Glow discharge is a plasma formed by the passage of electric
current through a gas. It is often created by applying a voltage between two electrodes in a
glass tube containing a low-pressure gas. When the voltage exceeds a value called the striking
voltage, the gas ionization becomes self-sustaining, and the tube glows with a colored light.
The color depends on the gas used (commonly you use air).
When you are doing SEM, you always coat your sample.
The plasma can cause Sputtering: atoms ejected from
solid material. Generated by bombarding the surface with
energetic particles of a plasma or gas. Can be used for
depositing a thin layer (used for SEM sample
preperation).
Sample preperation considerations
Sample protection within the TEM electrons are accelerated in high vacuum and have
high energy damaging for biological samples.
Sample thickness The sample needs to be thin enough for electrons to go through (< 1
um). The penetration depth of electrons increases with increasing acceleration voltage.
Sample contrast biological samples mainly consist of light elements (H, C, N and O)
which weakly scatter electrons and hence show poor contrast.
