Welcome to the soft materials and cryo-TEM section of the nanotechnologies course.
My name is Michelle Plue.
In another video, we discussed how
the Transmission Electron Microscope or TEM works and how to image hard material samples.
In this lesson, we're going to talk about how to view soft materials using the TEM.
How to prepare these types of samples, cryo-TEM and safety.
We will then image soft materials and cryo-TEM samples.
The first section of this video will cover what
soft materials are and how to prepare them for the TEM.
We will then discuss a little about cryo-TEM and how to prepare those samples.
Soft materials are flexible substances like polymers,
biological in nature, or are contained in a liquid.
Some examples of soft materials are things like paper,
fabrics, plants, animal tissue, plastics and cells.
These samples range in size from tenths of nanometers, like a virus,
to things that are many microns in size,
like bacteria or cells,
to things that are many millimeters in size,
like fabric and paper.
Bacteria are about 1,000 times larger than some viruses.
One way to think about the difference in size between a bacteria and
a virus would be like comparing a football field to a piece of notebook paper.
In this case the size of the bacteria,
which is our football field,
is really only one tenth of the diameter of a human hair.
The TEM is used to see the inside of soft materials.
Have you ever seen a picture of a mitochondria or a nucleus?
That was probably imaged with a TEM.
To image the inside of samples,
they must be prepared in very thin slices called thin sections.
Thin sections of the samples are only 70 to 90 nanometers thick.
To slice soft samples this thin,
we must prepare them and harden them.
Let's talk about preparing the soft samples to be imaged.
There are a few different ways to prepare soft materials for imaging.
One way to prepare them is to place them in epoxy resin.
The samples that do not contain water like fabrics and paper,
can be placed directly into the resin.
Samples that contain water,
like animal tissue or cells,
must have the water removed before they can be placed in the resin.
This process is called dehydration.
To dehydrate the samples,
we use a series of ethanol washes that replaces the water with the ethanol.
Once we have reached 100% ethanol we can place the samples into the resin.
The resin is then hardened in an oven.
We use an instrument called an ultra microtome that
has a diamond knife to make the thin sections.
The thin sections are then placed on a TEM grid.
These grids are then placed into the TEM for imaging.
cryo-TEM is another way to prepare soft material samples.
cryo-TEM is used when removing the water from the sample will change their shape or size.
These changes are called dehydration artifacts.
Sometimes, freezing samples will also give us better resolution.
We can use the better resolution to do a 3D reconstruction of the sample.
cryo-TEM involves freezing the sample that still has the water in it.
There are different ways to prepare cryo-TEM samples.
We will be talking about the plunge freezing method in this video.
Plunge freezing quickly dunks the sample into liquid ethane or propane to freeze it.
The reason we use liquid ethane or propane is because they
are the right temperature to get the right type of ice to form.
All ice is not equal.
There are three different types of ice that can form at atmospheric pressure.
Hexagonal ice, cubic ice, and vitreous ice.
Hexagonal ice is the ice we see every day.
It is the type of ice crystal that forms in an ice cube or a snowflake.
Cubic ice is formed when vitreous ice warms up.
I refer to that as bad ice when I see it in the TEM.
Vitreous ice has no regular crystal structure and is called amorphous.
This is the only type of ice that we can see through with the TEM.
This is the good ice.
Always wear the right type of safety gear when preparing samples.
For biological sample prep,
you will need to wear a lab coat,
safety glasses and nitrile gloves.
Liquid nitrogen is a cryogenic liquid.
It freezes water almost instantly.
Care must be taken when handling it.
See what happens when this rose is put in to liquid nitrogen.
When preparing cryo-TEM samples,
you will need to change your gloves.
Gloves become brittle when frozen and can freeze to your skin.
Now we're ready to prepare our samples.
