3.2.1 Studying cells

Question 1

What is magnification?

Answer 1

How much bigger the image is than the specimen

Magnification = image size / actual size

Question 2

What is resolution?

Answer 2

The shortest distance between two points on a specimen that can still be distinguished by the microscope camera as separate entities

Question 3

What is resolution limited by?

Answer 3

Wavelength

Question 4

How does an optical light microscope produce an image?

Answer 4

Using light rays that are transmitted through the specimen and two lenses

Question 5

What is the maximum magnification of an optical light microscope?

Answer 5

x 1 500

Question 6

What are the limitations of an optical microscope?

Answer 6

Low resolution - maximum about 0.2µm (long wavelength of light)
Cannot see cell ultrastructure smaller than 0.2µm: lysosomes, ribosomes, endoplasmic reticulum

Question 7

How does an electron microscope produce an image?

Answer 7

Using electrons to produce black and white images

Question 8

What is the maximum magnification of an electron microscope?

Answer 8

x 1 500 000

Question 9

What is the resolution of an electron microscope and why?

Answer 9

High resolution - maximum about 0.0002µm (x1000 light microscope) - electrons have very short wavelength
Can see more organelles

Question 10

What are the limitations of an electron microscope?

Answer 10

Process involves specimen preparation: staining with toxic metal, viewing in a vacuum, specimen cannot be alive
Colour is only added artificially
Specimen must be very thin - cut with microtome (TEM)
Artefacts could be present in photomicrograph from method of preparation

Question 11

What are the two types of electron microscope?

Answer 11

Transmission EM
Scanning EM

Question 12

How does a TEM work?

Answer 12

Electromagnets are used to focus a beam of electrons, which is then transmitted through the specimen

Denser parts of the specimen absorb more electrons - darker images

This helps to identify where organelles are located

Its high resolution allows you to see internal structure of organelles e.g. chloroplasts

Question 13

What are the limitations of TEM?

Answer 13

Specimen must be view in a vacuum, so movement living organisms could not be observed

They can only be used on very thin specimens (cut using a microtome) so that electrons could pass through them

Question 14

How does a SEM work?

Answer 14

A beam of electrons is scanned across the specimen, focused using magnetic condensers

Electrons are reflected off from the specimen and are gathered in a cathode ray tube to form an image

They produce 3D images

Question 15

What is cell fractionation?

Answer 15

The process of breaking cells and separating out different organelles within

Question 16

What is done before cell fractioning?

Answer 16

Homogenisation and fractionisation

Question 17

What happens in homogenisation?

Answer 17

Tissue is cut up and stored in cold, isotonic buffered solution

They are further cut up using a homogeniser

This releases organelles from the cell

Question 18

What happens after homogenisation?

Answer 18

Filtration

The resultant liquid from homogenisation is called the homogenate

It will then be filtered to remove any complete cells or large pieces of cell/tissue debris

Question 19

What happens after filtration?

Answer 19

Ultracentrifugation

Solution is poured into a tube and placed into a centrifuge

It will be spun first at a slower speed, which forces the heaviest organelles (nuclei) to the bottom of the tube - forming a pellet. The rest of the organelles stay suspended in the fluid above called the supernatant

The supernatant will be drained off and placed into another tube. This process is repeated at a higher speed and a new pellet and supernatant will form.

This process is repeated until the desired organelles are spun out with the centrifuge