3.2.1.3 Methods of studying cells

Question 1

What are the principles of optical microscopes?

Answer 1

Optical microscopes use visible light and lenses to magnify the image.

Question 2

What are the limitations of optical microscopes?

Answer 2

They have a low resolution due to the wavelength of light and cannot view structures smaller than 0.2 micrometres.

They also have a lower magnification compared to electron microscopes.

Question 3

What are the principles of transmission electron microscopes (TEM)?

Answer 3

TEMs use a beam of electrons transmitted through a thin specimen.

Electrons are focused using electromagnets to produce an image.

They provide high resolution and can view internal structures of organelles.

Question 4

What are the limitations of transmission electron microscopes (TEM)?

Answer 4

TEMs require a vacuum

Question 5

What are the principles of scanning electron microscopes (SEM)?

Answer 5

SEMs use a beam of electrons that scan the surface of the specimen. The electrons are reflected to form a 3D image of the surface structure.

Question 6

What are the limitations of scanning electron microscopes (SEM)?

Answer 6

SEMs have a lower resolution than TEMs. They also require a vacuum and cannot view live specimens. Artefacts may be introduced during preparation.

Question 7

Define magnification.

Answer 7

Magnification is how much larger the image of a specimen appears compared to its actual size.

Question 8

Define resolution.

Answer 8

Resolution is the ability to distinguish between two points that are close together as separate entities.

Question 9

What is the formula for calculating magnification?

Answer 9

Magnification = Image size / Actual size

Question 10

What are the principles of cell fractionation?

Answer 10

Cell fractionation involves breaking open cells to release organelles. The homogenate is filtered to remove debris and whole cells

Question 11

What are the steps of ultracentrifugation?

Answer 11

1. Homogenisation: Cells are broken to release organelles.
2. Filtration: Removes debris and whole cells.
3. Centrifugation: Spins at increasing speeds

Question 12

Why was distinguishing between artefacts and cell organelles a challenge?

Answer 12

• Artefacts are structures seen under the microscope that are not present in the cell.
• They are often caused by preparation techniques.
• Early microscopy techniques made it difficult to differentiate artefacts from genuine organelles.
• This required repeated observations and technological improvements.

Question 13

Microscopes - How to measure objects using an eyepiece graticule (3)

Answer 13

1. Use eyepiece graticule to measure the object e.g. nucleus or capillary
2. Calibrate eyepiece graticule against stage micrometer
3. Take a number of measurements and calculate the mean

Question 14

Microscopes - Advantages and Limitations of Transmission Electron Microscope (TEM)

Answer 14

Advantages:

1. Small objects can be seen;
2. TEM has high resolution as wavelength of electrons shorter;

Limitations:

1. Cannot look at living cells as cells must be in a vacuum;
2. Must be thin specimen;
3. Preparation may create artefact;
4. Does not produce colour image;

Question 15

Microscopes – Advantage of electron
microscope over optical microscope (2)

Answer 15

1. High resolution;
2. Can see internal structure of organelles

Question 16

Microscopes - Comparison of TEM and optical microscope (8)

Answer 16

1. TEM use electrons and optical use light;
2. TEM allows a greater resolution;
3. (So with TEM) smaller organelles/named cell structure can be observed
4. TEM view only dead/dehydrated specimens and optical (can) view live specimens;
5. TEM does not show colour and optical (can);
6. TEM requires thinner specimens;
7. TEM requires a more complex/time consuming preparation;
8. TEM focuses using magnets and optical uses (glass) lenses;

Question 17

Microscopes - The resolution of an
image obtained using an electron
microscope is higher than the
resolution of an image obtained using an optical microscope.

Explain why. (2)

Answer 17

Shorter wavelength between electrons; OR Longer wavelength in light rays;

Question 18

Microscopes - Describe and explain one difference between TEM and SEM (2)

Answer 18

1. 3D image (with SEM), not 2D image
   OR
   Lower resolution (with SEM)
   OR
   (Only) surface visible with SEM, but internal structures visible with TEM;
2. (Because) electrons deflected/bounce off (using SEM) OR Electrons transmitted/pass through (using TEM);

Question 19

Homogenisation – Conditions required for cell homogenisation (3)

Answer 19

1. Ice-cold – Slows/stops enzyme activity to prevent digestion of organelles/mitochondria;
2. Buffered – Maintains pH so that enzymes/proteins are not denatured;
3. Same water potential – Prevents osmosis so no lysis/shrinkage of organelles/mitochondria;

Question 20

Homogenisation & Ultracentrifugation – How to separate mitochondria? (4)

Answer 20

1. Break open cells/homogenise/produce homogenate;
2. Remove unbroken cells/larger debris by filtration;
3. Centrifuge highest density organelle nuclei obtained as pellet at slowest speed
4. Mitochondria in 2nd pellet as less dense than nucleus/organelle in first pellet;

Question 21

Suggest why scientists can use detergent to break open cells instead of homogenisation (2)

Answer 21

1. Cell membranes made from phospholipid;
2. (Detergent) dissolves membranes / phospholipid (bilayer);