Lecture 3 - electron microscopy

Question 1

What is wavelength and resolution for electron microscopy?

Answer 1

wavelength - 2.5pm

Resolution - 0.1nm

Question 2

What is the wavelength and resolution for light microscopy?

Answer 2

wavelength - 400 - 710 nm

resolution - 200 nm

Question 3

How do transmission electron microscopy work?

Answer 3

Electrons are emitted from a filament & accelerated in an electric field

Condenser lens focuses the electron beam

electrons either scatter or hit a fluorescent screen at the bottom of the microscope

Question 4

How do scanning electron microscopes work?

Answer 4

Electrons are emitted from a filament & accelerated in an electric field

Condenser lens focuses the electron beam

electrons are focused on a metal coted specimen, electrons from the metal are collected by a detector

Question 5

What are the 4 steps you have to do , to do a direct examination with transmission techniques?

Answer 5

• grids
• formvar
• contrast
• replication

Question 6

What is formvar in direct examination, for TEM?

Answer 6

• samples placed on a copper grid coated with formvar

Question 7

what are the for techniques for using direct examination for TEM microscopes?

Answer 7

• grids and formvar
• staining
• shadowing
• replication

Question 8

What are the two forms can you examine specimens for TEM?

Answer 8

• Direct

- sections from tissues

Question 9

Why do you have to stain TEM specimens for direct examination

Answer 9

• Biological materials are composed of C, H, O, N.

- Contrast is enhanced by staining with heavy metals such as lead, uranium , tungsten and gold

Question 10

What is shadowing for TEM specimens for direct examination?

Answer 10

Heavy metal is evaporated from a wire in a vacuum chamber casting a shadow on the adjacent sample

Question 11

Why do you have to make replicas of samples

Answer 11

if the sample is extremely fragile

Question 12

What do you have to do when using transmission EM techniques for sections from tissues?

Answer 12

• fixation
• dehydration
• embedding
• sectioning
• staining

Question 13

How can you fixate sections from tissues?

Answer 13

• use chemicals;
• Glutaraldehyde ( protein and amino acids)
• Osmum tetroxide (proteins and lipids)
• Potassium permanganate ( membranes)

Question 14

How can you dehydrate sections from tissues?

Answer 14

• 10% to 100% ethanol in a series of steps

Question 15

How do you embed sections from tissues?

Answer 15

Place sample in BEEM capsule
Infiltration with un-polymerised epoxy resin, Epon or Araldite
Polymerisation of resin in BEEM capsule
Remove resin block

Question 16

How do you section sections from tissues?

Answer 16

Ultra-microtome (estimate section thickness by interference colours).
Sections (50-90 nm thick) are picked up on formvar coated grids.

Question 17

What are the localisation techniques for light microscopy?

Answer 17

Histochemical dyes

Antibodies linked to fluorescein isothiocyanate (FITC)
Green

Fluorescent Protein (GFP)

Chromogenic enzyme substrate

Question 18

What are the localisation techniques for electron microscopy?

Answer 18

Antibody linked to colloidal gold. Different sizes can differentiate two different compounds simultaneously.

Enzyme localisation by linking product to heavy metal e.g. lipases with Pb.

Enzyme localisation if products are electron dense e.g. peroxidase

Question 19

What are the features of the scanning electron microscope?

Answer 19

Wide range of magnification x10 to x100,000
Great depth of focus (e.g. at x1000 depth of focus 10 μ, with light microscopy 0.6 μ)
High resolution <10 nm
Specimen can be rotated and tilted
Large irregular specimens can be viewed

Question 20

How do you prepare samples for SEM microscopy?

Answer 20

Similar to TEM, but robust specimens (pollen, diatoms, insects withstand vacuum without collapse)
Delicate specimens need to be dehydrated

Question 21

How do you perform critical point drying?

Answer 21

Dehydrate in ethanol-water series

Replace with amyl acetate
Subject to liquid CO2 under pressure

Raise temperature, liquid CO2 converts to gas leaving dry specimen