2.1 - Microscopy

Question 1

Describe how light microscopes work ?

Answer 1

• lenses focus rays of light and magnify the view of a thin slice of specimen
• different structures absorb different amounts and wavelengths of light
• reflected light is transmitted to the observer via the the objective lens and the eyepiece

Question 2

Describe how a transmission electron microscope ( TEM ) works

Answer 2

• pass a high energy beam of electrons through a thin slice of specimen
• more dense structures appear darker since they absorb more electrons
• focus image onto fluorescent screen it photographic plate using magnetic lenses

Question 3

Describe how a scanning electron microscope (sem) works

Answer 3

• Focus a beam of electrons onto a specimens surface using a electromagnetic lenses
• reflected electrons hit a Collecting device and are amplified to produce an image on a photographic plate

Question 4

Describe how a laser scanning confocal microscope works

Answer 4

• focus a laser beam onto a small area on a samples surface using objective lenses
• flurophores in the sample emit photons
• photomultiplier tube amplifies the signal onto a detector
• an image is produced pixel by pixel in the correct order

Question 5

State the equation to calculate the actual size

Answer 5

Actual size = image size / magnification

  I A       M

I = image size
A = actual size
M = magnification

Question 6

What is magnification?

Answer 6

The factor by which the image is larger than the actual specimen

Question 7

What is resolution?

Answer 7

The smallest separation distance at which 2 separate structures can be distinguished from one another

Question 8

Why do samples need to be stained for light microscopes ?

Answer 8

• colored dye binds to the structures
• facilities absorption of wavelengths of light to produce an image

Question 9

What is differential staining?

Answer 9

• The contrast between heavily and lightly stained areas distinguishes structures

Question 10

What is the magnification and resolution of a compound optical microscope?

Answer 10

Magnification = x2000
Resolution = 200nm

Question 11

State the magnification and resolution of a TEM

Answer 11

Magnification = x 500,000
Resolution = 0.5 nm

Question 12

State the magnification and resolution of an SEM

Answer 12

Magnification = x500,000
Resolution = 3-10 nm

Question 13

Explain how to use an eyepiece graticule and stage micrometer to measure the size of a structure?

Answer 13

1. Place a micrometer on stage to calibrate eyepiece graticule
2. Line up scales on the graticule and micrometer
3. Count how many graticule divisions are in 100um on the micrometer
4. Length of 1 eyepiece division = 100 um / number of divisions
5. Use the calibrated value to calculate the actual length of structures

Question 14

What is a electron microscope?

Answer 14

• uses electrons to form an image
• greatly increases the resolution - more detailed image
• a beam of electrons has a smaller wavelength than light so thus can resolve two objects that are extremely close together
• eg. TEM and SEM

Question 15

What is a advantage of TEM microscopes?

Answer 15

high resolution which allows the internal structures to be seen

Question 16

What is a disadvantage of TEM microscopes ?

Answer 16

• used with thin specimens
• cannot observe live specimen as there is a vacuum inside the TEM and all water must be removed from the specimen
• lengthy treatment - required to prepare specimens means that artefacts can be introduced
• do not produce colored images

Question 17

What is a advantage of SEM microscopes ?

Answer 17

• can be used on thick or 3D specimens
• allow the external 3D structure of specimens to be observed

Question 18

What’s a disadvantage of SEM microscopes ?

Answer 18

• lower resolution images
• cannot be observed to observe live specimens
• do not produce a colour image

Question 19

What’s a advantage of a Laser scanning confocal microscope?

Answer 19

• used on thick 3D or thick specimens
• allow external 3D structure to be observed
• high resolution due to the fact that the laser beam can be focused at very specific depths

Question 20

What’s a disadvantage of a laser scanning confocal microscope?

Answer 20

• slow process takes a long time to obtain an image
• laser has potential to cause photodamage to the cells

Question 21

What are common dyes used for staining a slide , and what colour do they turn cells ?

Answer 21

• toluidine turns cells blue
• phloroglucinol turns cells red/ pink

Question 22

Why do we stain slides ?

Answer 22

Staining provides a contrast to distinguish between different structures in a sample

Question 23

What is differential staining?

Answer 23

•When multiple stains are used and each stain binds to a specific cell structure
• staining each structure differently is important so that structures can be easily identified

Question 24

How do you view a sample under a light microscope?

Answer 24

1. Clip the slide onto the stage
2. Select the lowest powered objective lens
3. Use the coarse adjustment knob to move the objective lens to just above the slide
4. Adjust the focus by moving the lens away from the slide using the fine adjustment knob until a clear image appears
5. If a higher magnification is required use a high powered objective lens and refocus