Lesson 2 - Microscopy

Question 1

Magnification definition:

Answer 1

How many times larger the image is than the actual size of the object being viewed.

Question 2

How to see details?

Answer 2

Magnification does not change the visibility (details) of the image. Resolution is needed.

Question 3

Resolution definition:

Answer 3

The ability to see individual objects as separate entities.

Question 4

What limits resolution?

Answer 4

Diffraction

Question 5

Diffraction definition:

Answer 5

The tendency of light waves to spread as they pass close to physical structures such as those present in specimens being studied. The structures present in specimens are very close to each other and the light reflected from individual structures can overlap due to diffraction. This means that structures are no longer seen as separate entities and detail is lost.

Question 6

Optical microscopy structures:

Answer 6

If structures are closer than half a wavelength of light, then structures can not be seen individually.

Question 7

How to increase resolution?

Answer 7

Electron microscopy

Use beams of electrons, which have a wavelength a thousand times shorter than light.
- Electron beams are still diffracted but the shorter wavelength means that beams can be much closer before they overlap.
- This means that structures which are smaller and much closer together can be seen separately without diffraction blurring the image.

Question 8

Magnification equation?

Answer 8

Magnification = Size of Image / Actual size of object

Question 9

Limiting factor of light microscopy?

Answer 9

Resolution

Question 10

Electron microscopy?

Answer 10

• A beam of electrons with a wavelength of less than 1mm is used to illuminate the specimen.
• More detail of cell ultra structure can be seen because electrons have a much smaller wavelength than light.
• They produce images with a magnification of up to x 500,000 and still have clear resolution.

Question 11

Disadvantages of electron microscopy?

Answer 11

• Expensive pieces of equipment
• Can only be used in a carefully controlled environment in a dedicated space.
• Cells can be damaged by the electron beams
• The preparation process is complicated and there is the complication of artefacts (structures that are created in the preparation process). As techniques improve they can be eliminated.

Question 12

The two types of electron microscopes?

Answer 12

• Transmission electron microscope (TEM)
• Scanning electron microscope (SEM)

Question 13

Transmission Electron Microscope (TEM):

Answer 13

• Beam of electrons is transmitted through a specimen and focused to produce an image.
• This is similar to light microscopy
• Best resolution (with resolving power) of 0.5 nm

Question 14

Scanning electron microscope (SEM):

Answer 14

• Beam of electrons sent across the surface of a specimen and reflected electrons are collected.
• Resolving power is from 3 - 10 nm
• Resolution is not as good as transmission electron microscope
• 3D images created, giving valuable information about the appearance of structures.

Question 15

Preparation for electron microscopy?

Answer 15

• Fixation using chemicals or freezing
• Staining with heavy metals
• Dehydration using solvents

TEM: Set in resin and may be stained again

SEM: Fractured to expose the inside. Then stained with heavy metals again.

Question 16

Why are specimens prepared specifically for electron microscopes?

Answer 16

The inside of the microscope is a vacuum.

Question 17

Resolving power of light vs electron microscopes?

Answer 17

Light: 200 nm
Electron: 0.5 nm / 3-10 nm

Question 18

Example of artefact in light microscopy?

Answer 18

Air bubbles under cover slip

Question 19

What was the name given to the artefact thought to be an inward fold of the cell membrane in prokaryotic cells?

Answer 19

Meosome

Question 20

What did scientists believe the meosome was for?

Answer 20

Surface area of the folded surface was believed to be an important site of process of oxidative phosphorylation.

Question 21

How was the meosome disproved?

Answer 21

A non-chemical technique was developed called cryofixation. Mesosomes were no longer visible.

Question 22

Laser Scanning confocal microscopy:

Answer 22

A higher light intensity is used to illuminate specimens treated with chemical fluorescent dye.
Fluorescence is the absorption and re-radiation of light. Light of a longer wavelength and lower energy is emitted to produce magnified image.

Question 23

How the confocal microscope works?

Answer 23

Moves a single spot of focused light across a specimen. This causes flourescence from the components labelled with a dye. The emitted light from the specimen is filtered through a pinhole aperture.

Question 24

What light is detected?

Answer 24

Only light radiated from very close to the focal plane (the distance that gives the sharpest focus) is detected.
Light emitted from other parts of the specimen would reduce the resolution and cause blurring. Unwanted radiation does not pass through the pinhole and is not detected.

Question 25

Uses of laser scanning confocal microscopy?

Answer 25

Non-invasive and is currently used on diagnostics of diseases of the eye. Being developed for use in endoplasmic procedures. The fact that is can be used to see the distribution of molecules means that is it used in drug development.

Question 26

Future uses of laser scanning confocal microscopy?

Answer 26

Virtual biospies
- Especially for cases of suspected skin cancer

Question 27

Beamsplitter: in confocal microscope

Answer 27

Dichroic mirror, which only reflects one wavelength (from the laser) but allows other wavelengths (from sample) to pass through.

Question 28

Why are the pinholes positioned where they are in confocal microscope?

Answer 28

Light waves from the laser (illuminating the sample) follow the same path as the light waves radiated when sample flouresces. This means that they both have the same focal plane, hence the term confocal.

Question 29

How is high resolution obtained in confocal microscope?

Answer 29

Thin specimins of the sample are examined and light from elsewhere is removed.

Question 30

Confocal micrsocope: different dimensions?

Answer 30

Spot illuminating specimin is moved and two dimensional image produced.
Three dimensional image can be produced by creating images at different focal planes.