Lecture 2 - Electron Microscopy

Question 1

What are the wavelength of an electron microscope

Answer 1

~0.004nm
(shorter wavelengths have higher energy)

Question 2

What is the resolution of electron microscope

Answer 2

~0.2nm

Question 3

What is the magnification of an electron micrscope

Answer 3

x1000 - x200,000

Question 4

How does electron microscopy work

Answer 4

• Electrons are emitted from a filament & accelerated in an electric field
• Condenser lens focuses the electron beam
• TEM: Electrons either scatter or hit a fluorescent screen at the bottom of the microscope
• SEM: Electrons are focused on a metal coated specimen, electrons from the metal are collected by a detector
• Colum is maintained at very high vacuum

Question 5

What is fixation

Answer 5

• Biological fine structure needs to be preserved during sample presentation, so chemicals are used to prevent degradation

Question 6

What are some chemicals used in fixation

Answer 6

Gluteraldehyde (protein amino groups) covalently cross links proteins to neighbours
- Osmium tetroxide (proteins and lipids) – Stabilises lipid bilayers and proteins
- Potassium Permanganate KMnO4 – (membranes)

Question 7

How does dehydration occur

Answer 7

Dehydration using 10% to 100% ethanol series in 10% steps with very gentle agitation

Question 8

How does embedding take place

Answer 8

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

Question 9

How does sectioning occur

Answer 9

• Cut with microtome (diamond usually)
• Put on EM grid

Question 10

What are artefacts

Answer 10

• Look like part of the microscope sample but are actually a side-effect of sample preparation or the conditions in the microscope

Question 11

Why is rapid freezing good for microscopy

Answer 11

• If aqueous system cooled fast enough, ice crystals do not form
• Some specimens can be examined directly
• To look at thin sections need to replace water with organic solvents, embed in resin, section etc…

Question 12

How can image clarity be improved and what is it dependant on

Answer 12

Image clarity depends upon having a range of contrasting electron densities in the specimen

Tissues often impregnated with heavy metals (Osmium, Uranium, Lead) to provide contrast

Question 13

What is Tomography

Answer 13

Serial sections can be used to reconstruct 3D image, usually an organelle (eg. Golgi apparatus below), colour is added due to false colouring on a computer

Question 14

How are cell components localised by EM (page 5 notes)

Answer 14

• Antibody linked to colloidal gold – different sizes can differentiate two different compounds simultaneously
• Enzyme localisation by linking product to heavy metal eg lipases with Lead
• Enzyme localisation if products are electron dense eg. Peroxidase

Question 15

What are some features of SEM

Answer 15

• 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 16

What are some features of SEM imaging

Answer 16

• Directly produces image of surface of a specimen
• Specimen is fixed dried and coated with a thin layer of heavy metal
• Specimen scanned with thin beam of electrons
• Scattered electrons detected
• 3D image of surface created.