2.3 More Microscopy

Question 1

Describe light microscopes

Answer 1

• They use light to illuminate the specimen.
• they have a lower resolution than electron microscopes
• have a maximum resolution of about 0.2 micrometers
• usual lay used to look at whole cells or tissues
  Maximum useful magnification of a light microscope is x1500

Question 2

Describe laser scanning confocal microscopes

Answer 2

• these are a special type of light microscope that use laser beams to scan a specimen thats usually tagged with fluorescent dyes
• a laser beam is focused through a lens which is aimed at a beam splitters. This splits the beam and some of the light is directed to the specimen
• when the laser hits the dyes it causes them to give off fluorescent dyes
• this light is then focused through a pinhole onto a detector. The detector is hooked up to a computer which generates an image
• the pinhole means that any out of focus light is blocked out in order to produce a much clearer image
• these microscopes can be used to look at objects at different depths in thick specimens
• multiple images produced by the microscope can be combined by the computer to produce a 3D images

Question 3

What are electron microscopes and what are the two types?

Answer 3

• electron microscopes use electrons instead of light to form an image. They have a higher resolution than light microscopes so give more detailed images
• transmission electron microscope (TEM) and scanning electron microscope (SEM)

Question 4

Describe TEMs

Answer 4

• a beam of electron is transmitted through a specimen and focused to produce an image
  Best resolving power 0.5 nanometers

Question 5

Describe SEMs

Answer 5

A beam of electrons is sent across surface of a specimen and reflected electrons are collected to produce 3D images
Resolving power 3-10nm

Question 6

What are artefacts?

Answer 6

• an artefact is a visible structural detail caused by processing specimen and is not a feature of the specimen
• e.g. the bubbles that get trapped under cover slip as you prepare a slide for light microscopy are artefacts

Question 7

How do you prepare a sample for electron microscopes?

Answer 7

Inside of an electron microscope is a vacuum to ensure electron beams travel in straight lines. Therefore preparation:
Involves fixation of chemical or freezing, staining with heavy metals and dehydration with solvents