Module 2 - Methods of studying cells

Question 1

How do optical light microscopes work?

Answer 1

1) Light passes through the specimen
2) Into the condenser lens
3) Into the objective viewing lens
4) Into observers eye where an image in the brain is formed

Question 2

Limitations of optical light microscopes?

Answer 2

A low resolution so cannot see smaller organelles such as ER, Low resolution so won’t be a clear image, 3D images not produced

Question 3

What is resolution?

Answer 3

the minimum distance at which two distinct points of a specimen appear seperate.

Question 4

What is a TEM microscope? How does it work?

Answer 4

A transmission electron microscope uses electromagnets to focus a beam of electrons. Denser parts absorb electrons this is why they appear darker on final image.

Question 5

Advantages of TEM

Answer 5

High resolution, High magnification producing detailed images of small organelles.

Question 6

Limitations of TEM

Answer 6

organisms must be dead, thin piece has to be used, real colour not shown, artefacts possible, doesn’t produce 3D images

Question 7

What is a SEM microscope? How does it work?

Answer 7

SEMs scan a beam of electrons across the specimen

This beam bounces off the surface of the specimen and the electrons are detected, forming an image.

Question 8

Advantages of SEM

Answer 8

high magnification, 3D images are produced, live specimen can be used

Question 9

Limitations of SEM

Answer 9

real colour cannot be seen, artefacts possible, lower resolution than TEM but higher than light.

Question 10

Why are electron microscopes better than light microscopes?

Answer 10

Higher resolution due to electrons being smaller than light photons.

Question 11

What is the formula for magnification?

Answer 11

Magnification = image size / actual size

Question 12

What is cell fractionation? What are the 2 stages?

Answer 12

The process where cells are broken apart and different organelles within them are separated out. Homogenisation and ultracentrifugation.

Question 13

What is the first stage homogenisation?

Answer 13

Homogenisation consists of using a homogeniser to break open the cells by breaking the plasma membrane releasing organelles into a solution called homogenate.

Question 14

What conditions does the solution that we place the homogenised cells need to be?

Answer 14

Isotonic to prevent damage to (the given cell) from water moving into the cell via osmosis (maintains same water potential), buffered prevents proteins from becoming denatured and cold to reduce the activity of enzymes.

Question 15

Explain the stage after homogenisation - filtration

Answer 15

Filtered through a gauze to remove large debris or whole cells leaving a filtrate.

Question 16

Explain the last stage; ultracentrifugation

Answer 16

A centrifuge is used to spin the filtrate at different speeds. It starts with the lowest speed which forms a pellet containing the heaviest organelle, the supernatant is the remaining organelles above. The supernatant is dispersed into a separate test tube and spun at a faster speed separating the next heaviest organelles. This is repeated

Question 17

What is the order of mass of organelles?

Answer 17

Nuclei, chloroplasts (if its plant tissue), mitochondria, lysosomes, ER and ribosomes

Question 18

What is an eyepiece graticule?

Answer 18

A glass piece of equipment that you place on an eyepiece to measure the object

Question 19

Why do we use an eyepiece graticule and why is it important to calibrate?

Answer 19

To measure the size of an object. Calibrating is important because each objective lens shows a different magnification.

Question 20

How do we calibrate an eyepiece?

Answer 20

Line up one of the divisions on the eyepiece graticule with a fixed point on the stage micrometre. Count the number of divisions on the eyepiece graticule that correspond with a set measurement on the stage micrometre. Calculate the distance in micrometres of one division on the eyepiece graticule.

Question 21

What is the formula for finding out what 1 graticule division is?

Answer 21

1 graticule division = number of micrometres ÷ number of graticule division