2.3- more microscopy

Question 1

What is electron microscopy?

Answer 1

In electron microscopy, a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen.

• More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves.
• they can produce images with magnifications of up to x500000 and still have a clear resolution.

Question 2

What are the two types of electron microscopy?

Answer 2

Transmission electron microscope (TEM):
-a beam of electrons is transmitted through the specimen and then focused to produce an image.
similar to light microscopy.
Best resolution with a resolving power of 0.5nm

Scanning electron microscope (SEM):
- beam of electrons sent across the surface of a specimen and the reflected electrons are collected. Three dimensional images of surfaces are produced.
Resolving power is from 3-10nm, so resolution is not as good as TEM.

Question 3

Explain sample preparation for electron microscopes.

Answer 3

Involves:

• Fixation using chemicals/ freezing.
• Staining with heavy metals.
• Dehydration with solvents

Then:
TEM = set in resin and stained again.
SEM= fractured to expose insides and coated with heavy metals.

Question 4

How does a laser scanning confocal microscope work?

Answer 4

1. Moves a single spot of focused light across the specimen. (Point illumination)
2. causes fluorescence ( the absorption and re radiation of light) from the components labelled with a ‘dye’
3. Emitted light is filtered through a pinhole aperture. Only light radiated from close to the focal point is detected.

Question 5

Other key facts about laser scanning confocal microscopes.

Answer 5

• unwanted radiation would cause blurring and reduce the resolution so it doesn’t pass through the pinhole and therefore isn’t detected.
• thin sections of specimen examined = allows high resolution images to be obtained.
• 2D and 3D images can be formed.
• non-invasive: used in the diagnosis of diseases of the eye.
• position of the two pinholes means the light waves from the laser follow the same oath as the radiated light waves from the sample = same focal place (confocal)

Question 6

What is resolution, how is it limited and how can it be increased?

Answer 6

resolution is the ability to see individual objects as separate entities.

• Limited by the diffraction of light as structures are very close and thus causes light to overlap and as a result detail is lost.
• Increased by using beams of electrons as they have a shorter wavelength.