Electron microscopy

Question 1

How does the resolution in electron microscopy compare to light microscopy?

Answer 1

Light: 200nm, electron: 0.1nm.

Question 2

What is the basis of the electron microscope?

Answer 2

Electrons are emitted from a filament and accelerated in an electric field. A condenser lens focuses the electron beam.

Question 3

How do TEM and SEM differ?

Answer 3

In TEM, electrons scatter or hit a fluorescent screen at the bottom of the microscope, whereas in SEM electrons are focused on a metal coated speciment and the electrons are then collected by a detector. The electrons are already scattered before they hit the specimen in SEM.

Question 4

Why can specimens not be alive in electron microscopy?

Answer 4

Due to the high pressure vacuum that needs to be present.

Question 5

What method is used to view samples in TEM?

Answer 5

A transparent formvar film is floated off a glass slide onto the surface of water in a petri dish. Grids at the bottom of the fish are lifted out of the water and the film will be deposited on them.

Question 6

Why is a support film needed in TEM?

Answer 6

To enhance stability and conductivity when exposed to the electrons.

Question 7

How can sample contrast be increased in TEM?

Answer 7

Negative staining using heavy metals such as lead, uranium, tungsten and gold. Shadowing can be used where heavy metal is evaporated from a wire in a vacuum chamber to cast a shadow on the adjacent sample.

Question 8

How does shadowing work?

Answer 8

The sample is spread on a mica surface and then dried. The sample is coated with a film of heavy metal and the remaining biological material is then dissolved with acid and bleach.

Question 9

How can protein amino groups be preserved during sample preparation?

Answer 9

Using glutaraldehyde.

Question 10

How can proteins and lipids be preserved during sample preparation?

Answer 10

Using osmium tetroxide.

Question 11

How can membranes be preserved during sample preparation?

Answer 11

Using potassium permanganate.

Question 12

Why do samples need to be preserved in sample preparation?

Answer 12

As samples are usually around 70% water and they would boil in the EM vacuum.

Question 13

How can the samples be dehydrated?

Answer 13

Using increasing concentrations of ethanol in different steps.

Question 14

What is the purpose of embedding in TEM?

Answer 14

To produce blocks that are suitable for ultra-thin sectioning and still preserve the fine structure of the specimen.

Question 15

How can samples be embedded?

Answer 15

The sample should be placed in a BEEM capsule and infiltrated with un-polymerised epoxy resin, Epon or Araldite. The resin is them polymerised in the capsule and the resin block should be removed.

Question 16

How can tissues be sectioned?

Answer 16

Using an ultra-microtome to produce ultra-thin sections (50-90nm thick). These are then picked up on formvar coated grids.

Question 17

How do fixatives differ?

Answer 17

They can vary in their rate of penetration, their ability to fix different molecules and can cause shrinkage or swelling of tissues.

Question 18

How can cell components be localized in light microscopy?

Answer 18

Using histochemical dyes, antibodies linked to fluorescein isothiocyanate, GFP and chromogenic enzyme substrate.

Question 19

How can cell components be localized in electron microscopy?

Answer 19

An antibody linked to colloidal gold, enzyme localization by linking product to heavy metal e.g lipases with Pb and enzyme localization if products are electron dense e.g. peroxidase.

Question 20

How can proteins be detected in electron microscopy?

Answer 20

Antibodies and Protein A-gold can be used. Protein A-gold will bind to the antibodies added and can locate teh molecule of interest.

Question 21

How can different sizes of colloidal gold be used to identify different molecules?

Answer 21

The different sizes can be used to identify different molecules simultaneously.

Question 22

What can be used to identify phenols?

Answer 22

Peroxidase - it converts phenols to electron dense products.

Question 23

How does SEM work?

Answer 23

Electron beams scan the specimen and cause it to emit electrons to give a detailed image.

Question 24

What is the magnification range of SEM?

Answer 24

x10 to x100000

Question 25

What is the resolution of SEM?

Answer 25

<10nm

Question 26

What are some other key features of SEM?

Answer 26

There is a great depth of focus, the specimen can be rotated and tilted and large, irregular specimens can be viewed.

Question 27

What kind of images are produced with SEM?

Answer 27

3D images that can be artificially coloured.

Question 28

What is the preparation for samples in SEM?

Answer 28

Similar to TEM, but delicate specimens need to be dehydrated.

Question 29

What is the process for critical point drying?

Answer 29

The species needs to be dehydrated in an ethanol-water series, replaced with amyl acetate and subjected to liquid CO2 under pressure and then raise the temperature as liquid CO2 will convert to a gas to leave a dry specimen.

Question 30

What are the problems with critical point drying in SEM?

Answer 30

There may be shrinkage or the removal of substances in organic solvents such as leaf wax.

Question 31

How can the problems with critical point drying in SEM be overcome?

Answer 31

Cryo-SEM.

Question 32

What does Cryo-SEM mean?

Answer 32

Low temperature scanning electron microscopy.

Question 33

Why is cryo-SEM favoured over the normal SEM?

Answer 33

The sample retains water as it is flash freezed in liquid nitrogen. There is no exposure to fixatives or solvents, and the preparation time is short.

Question 34

What are the steps in cryo-SEM?

Answer 34

Mount the specimen on a holder, flash freeze in liquid nitrogen, transfer to a vacuum chamber of SEM (-100C), warm the stage above -96C to sublimeaway any surface ice crystals and then coat with metal and observe the specimen.