2.1.1c Microscopy

Question 1

What is magnification

Answer 1

How many times bigger the image produced by the microscope is than the real life specimen

Question 2

What is resolution

Answer 2

The ability to distinguish between 2 objects that are close together (ability to separate 2 objects/see more detail)
How detailed the image is

Question 3

How to calculate magnification of a light microscope

Answer 3

Total magnification = Eyepiece lens magnification (10) x Objective lens magnification

Question 4

Magnification equation

Answer 4

IAM triangle

           Image size 

Actual size Magnification

Question 5

See images for labelled diagram of microscope

Question 6

Description of Light microscope

Answer 6

• Use light to form an image
• Lower resolution than electron microscopes (max of 0.2pm) so usually used to view eukaryotic cells (sa nuclei & possibly mitochondria & chloroplasts)
• This is bc it is difficult to distinguish between (resolve) two objects that are closer than the wavelength of light (500-650nm)
• Maximum useful magnification is x1500

Question 7

Description of Electron microscope

Answer 7

• Use electrons to form an image
• Higher resolution than light microscopes - so give more detail bc a beam of electrons has a much smaller wavelength than light.
• Maximum resolution of 0.2nm (1000x greater than optical microscopes) so is able to resolve 2 objects that are extremely close tg
• Can observe small organelles sa ribosomes, ER & lysosomes
• 2 types of e microscopes: TEM & SEM

Question 8

Advantages of Light microscope

Answer 8

• inexpensive to buy & operate
• small & portable
• simple sample preparation
• vacuum is not required
• natural colour of sample is seen (or stains are used)
• specimens can be living or dead

Question 9

Disadvantages of Electron microscope

Answer 9

• expensive to buy & operate
• large & needs to be installed
• complex sample preparation which often distorts material
• vacuum is required
• black & white images produced (can only be coloured digitally)
• specimens must be dead

Question 10

Electron microscopes: What are Scanning electron microscopes

Answer 10

• SEMs scan a beam of electrons across the surface of the specimen. This bounces off the surface & knocks off electrons from the specimen, which are detected in a cathode ray tube forming an image.
• SEMs therefore form 3D images that show the surface of a specimen
• Magnification of x500,000 or less

Question 11

Electron microscopes: Advantages of Scanning electron microscope

Answer 11

• Can be used on thick or 3D specimens
• allow external 3D structure to be observed

Question 12

Electron microscopes: Disadvantages of Scanning electron microscope

Answer 12

• Lower resolution than TEMs
• Cannot be used on live specimen (unlike optical microscopes)
• Do not produce a colour image (unlike optical microscopes)

Question 13

Electron microscopes: What are Transmission electron microscopes

Answer 13

• TEMs use electromagnets to focus a beam of electrons, which is transmitted through the specimen
• Denser parts of the specimen absorb more electrons, making them appear darker on the final image

Question 14

Electron microscopes: Advantages of Transmission electron microscope

Answer 14

• Provide high resolution images, so great detail of a range of organelles
• Internal structures can be seen
• High magnification of x1,000,000 or more

Question 15

Electron microscopes: Disadvantages of Transmission electron microscope

Answer 15

• Can only be used w very thin specimens.
• Cannot be used to observe live specimens bc of the vacuum inside the TEM. Plus all the water must be removed from the specimen
• Lengthy treatment required to prepare specimens. Artefacts could be introduced - these look like real structures but are acc the results of preserving & staining
• Do not produce a colour image & are in 2D

Question 16

What are Laser scanning confocal microscopes

Answer 16

• relatively new tech
• cells are viewed and stained w fluorescent dyes
• a thick section of tissue or small living organisms are scanned w a laser beam (focused light)
• the laser beam is reflected by the fluorescent dyes
• multiple depths of tissue section/organisms are scanned to produce an image. As if the laser beam is building the image layer by layer

Question 17

Advantages of Laser scanning confocal microscopes

Answer 17

• used on thick or 3D structures
• external 3D structure is observed
• very clear, high resolution bc the laser beam can be focused at a very specific depth. Cytoskeleton can be observed

Question 18

Disadvantages of Laser scanning confocal microscopes

Answer 18

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

Question 19

What is significant about tissues used in microscopy being transparent

Answer 19

Many tissues used in microscopy are transparent, letting light & electrons through them
- this makes it difficult to see details
- stains are often used to colour the tissues

Question 20

Staining for light microscopy

Answer 20

For light microscope, coloured dyes are used
- The stain is taken up by some parts of the object more than others - the contrast makes the different parts show up
- Specimens absorb specific colours of light whilst reflecting others, making structures visible
- Diff stains are used to make diff things show up (eosin used for cytoplasms, methylene blue for DNA)
- More than 1 stain can be used at once

common stains: methylene blue and eosin

Question 21

Staining for electron microscopy

Answer 21

For e microscopes, specimen must be stained to absorb/scatter the electrons
- electrons have no colour so dyes cause tissues to show up black/grey
- objects are dipped in solution of heavy metal compounds (like lead). The metal ions scatter the electrons, creating contrast - some parts of object shoe up darker than others
- colours are often added to image using image processing software

Question 22

Converting units

Answer 22

nm
/1000
μm
/1000
mm
/10
cm
/100
m
/1000
km

Question 23

What is the magnification of the eyepiece lens

Answer 23

x10

Question 24

How to deal with scale bars (microscopes calc)

Answer 24

1. measure actual length of scale bar w ruler (convert from cm to mm and then to μm)
2. divide by length it represents
3. this gives the magnification

Question 25

What is differential staining

Answer 25

A technique which involves many chemical stains being used to stain different parts of a cell in different colours, ensuring various parts are visible

Question 26

Name 2 positively charged stains commonly used

Answer 26

Crystal violet or Methylene blue are positively charged, and therefore are attracted to and stain negatively charged materials

Question 27

Name 2 negatively charged stains commonly used

Answer 27

Nigrosin and Congo reds are negatively charged, and therefore cannot enter the cells bc cytosol repels them. This creates a stained background and the unstained cells then stand out

Question 28

What is Gram staining

Answer 28

Gram staining, to visualise different bacteria, is another common use of differential staining. Two different stains are used: crystal violet and safranin

Question 29

Do chloroplasts need staining

Answer 29

NO

Question 30

2 ways of preparing a microscope slide

Answer 30

- Dry mount - Wet mount

Question 31

Preparing microscope slide: **Dry mount**

Answer 31

- The specimen needs to let light through it in order to be seen clearly (so make specimen as thin as possible) - Use tweezers to pick up specimen & put it in the middle of a clean slide - Put a cover slip (square of thin, transparent glass) on top

Question 32

Preparing microscope slide: **Wet mount**

Answer 32

- Start by pipetting small drop of water onto slide. Then use tweezers to place specimen on top of water drop - To put cover slip on, stand the slip upright on the slide, next to the water droplet. Carefully tilt & lower it so it covers the specimen - (try not to get any air bubbles under as may obstruct your view) - Once cover slip is on, put a drop of stain next to one edge of the cover slip. Then put paper towel next to the opposite edge - the stain will get drawn under the slip, across the specimen

Question 33

Steps on how to use a light microscope

Answer 33

1. Clip the **slide** containing the specimen onto the **stage** 2. Select the **lowest-powered objective lens** (lowest magnification) 3. Use **coarse adjustment knob** to bring stage up to just below the objective lens 4. Look down eyepiece (containing the ocular lens). Use **coarse adjustment knob** to move stage downwards, away from the objective lens until the image is roughly in focus 5. Adjust the focus w the **fine adjustment knob**, until a clear image is obtained 6. If you need to see specimen w greater magnification, swap to a higher-powered objective lens & refocus