B2 - Microscopy

Question 1

What is a compound microscope and what are their benefits? (2)

Answer 1

Have 2 lenses: objective & eyepiece lens

objective & eyepiece configuration allows for higher magnification than in a simple light microscope.

Question 2

How thin must the specimen be for a light microscope and why?

Answer 2

5 micrometres
- very thin & transparent
- light won’t pass through = no visible detail if specimen is thick/opaque

Question 3

Why is a blue light filter used in light microscopy?

Answer 3

• improves resolution

Question 4

Why is staining used in light microscopy? (3)

Answer 4

1) Increases contrast, as most organelles are transparent so little light is absorbed
2) Easier to identify organelles
3) organelles more visible

Question 5

How do positive and negative stains work on different parts of the cell?

Answer 5

positive:
- attracted to negative cell components e.g. DNA
- stain cell components

negative:
- repelled by negative cytosol
- stains background of cell

Question 6

Give examples of different types of staining in light microscopy.

Answer 6

H&E (haematoxylin & eosin)

positive:
- methylene blue
- crystal violet

negative:
congo red

Question 7

What is gram staining?

Answer 7

separates bacteria into gram-positive and gram-negative.
- Apply crystal violet to bacteria on slide & wash w/ alcohol
- G+ retain stain = appear blue due to thicker peptidoglycan cell walls
= G- lose stain = appear red due to thinner peptidoglycan cell walls

Question 8

How is a dry mount prepared? (in detail)

Answer 8

tissue placed in fixative(formaldehyde), otherwise would decompose.
1) Dehydrate:
- replace H2O w/ alcohol as wax doesn’t mix with H2O
2) Place dehydrated tissue sample in metal mould & pour wax on.
3) Place plastic holder in mould & leave to cool
4) Lift plastic holder from mould & place wax tissue in a MICROTOME, which cuts 5micrometre slices
5) Place on slide for staining

Question 9

How is a squash slide prepared?

Answer 9

• wet mount squashed & cover slip placed on top
• good for soft tissue

Question 10

How is a wet mount prepared?

Answer 10

• specimen in liquid
• cover slip placed from an angle so no air bubbles

Question 11

How is a smear slide prepared?

Answer 11

• smear sample w/ edge of slide
• thin coat
• e.g. blood

Question 12

Define resolution.

Answer 12

The ability to distinguish two close together objects as separate structures.

Question 13

What are the limitations of resolution?

Answer 13

• Limited by diffraction (light waves spreading when close to physical structures)
• light reflected from close structures overlap, therefore not seen as separate objects
• structures closer than 1/2 the wavelength can’t be seen seperately.

Question 14

Why do electron microscopes have a higher resolution than light microscopes?

Answer 14

electron beams have a shorter wavelength than visible light

Question 15

What is the resolution and magnification of a light microscope?

Answer 15

resolution: 200nm
magnification: x1500 to 2000

Question 16

What is the resolution and magnification of a scanning electron microscope?

Answer 16

resolution: 2-10nm
magnification: x100,000 to 500,000

Question 17

What is the resolution and magnification of a transmission electron microscope?

Answer 17

resolution: 0.2-0.5nm
magnification: x500,000 to 2,000,000

Question 18

How is a sample prepared for an electron microscope?

Answer 18

• tissues fixed with glutaraldehyde
• dehydrated (H20 replaced w/ ethanol)
• tissue embedded in hard resin
• cut w/ diamond knife on an ultramicrotome
• stained w/ METAL

Question 19

Why are artefacts more common in electron miscroscopes?

Answer 19

• more complex preparation
• ## specimen may be damaged by electron beam

Question 20

What are the advantages of LSCM?

Answer 20

• visualise & watch specific proteins
• thick or 3D specimens

Question 21

What is LSCM?

Answer 21

• laser scanning confocal microscopy
• fluorescent microscope
• uses higher light intensity to illuminate specimen that’s been treated with fluorescent dye.

Question 22

Define fluorescence.

Answer 22

the absorption and re-radiation of light at a longer wavelength (hence diff colour)

Question 23

How can LSCM be used to tag & follow antibodies?

Answer 23

GFP (green fluorescent protein) produces green light when illuminated by UV. Attach GFP to antibody & locate protein it binds to.

Question 24

What is the focal plane in LSCM?

Answer 24

the distance from the specimen that gives the sharpest focus.

Question 25

Why is LSCM ‘confocal’?

Answer 25

• position of the 2 pinholes means that waves from the laser which illuminate the sample follow the same path as the light waves radiated when the sample fluoresces, therefore they both have the same focal plane.

Question 26

E xplain the steps of how to use a microscope (assume microscope has a 10x eyepiece lens). (7)

Answer 26

1) Place slide on stage
2) use lowest power (x4 objective lens initially, gives total magnification of x40)
3) switch light so that light passes through slide
5) move stage as close to lens as possible
6) slide must not touch lens as would crush slide
7) turn focussing knob to move slide away from lens and image comes into focus
8) Use fine focus to get clear image
9) repeat and refocus with x10 objective lens (x100 magnification) and x40 objective lens (x400 magnification)