Methods Of Studying Cells

Question 1

What are the 2 main microscopes

Answer 1

Optical light microscope + electron microscope

Question 2

How does optical light microscopy work?

Answer 2

Light passes through the pediment into the condenser lens of the microscope into the objective view lens and finally into the observer’s eye or connected to a computer

Question 3

What are some limitations of optical light microscopy?

Answer 3

• lower resolution so ribosomes, endoplasmic reticulums and lysosomes can’t be viewed properly
• low magnification - maximum x1500

Question 4

What are some principles of electron microscopy? 6

Answer 4

• 2 types: scanning and transmission
• use electrons to form images
• greater resolution - 0.002 micrometers
• maximum magnification x1’500’000
• black & white
• projected onto a fluorescent screen

Question 5

How does a transmission electron microscope work?

Answer 5

Projects an electron beam through a sample to form a 2D image

Question 6

What are some principles of the transmission electron microscope?

Answer 6

• produces very high resolution images
• 2D
• can produce very detailed images of cell organelles
• denser tissue appears darker since more electrons are absorbed

Question 7

What are some limitations of the transmission electron microscope?

Answer 7

• must be performed in a vacuum
• can’t visualise living material
• can only be used for thin tissues
• preparation takes time

Question 8

How does a scanning electron microscope work?

Answer 8

It directs an electron beam across a sample, scanning it and electrons released from the specimen are captured by a cathode ray tube

Question 9

What are some principles of the scanning electron microscope?

Answer 9

• can produce 3D images
• lower resolution than TEM
• scans the surface and captures the texture so can be used on thick specimens

Question 10

What are benefits of light microscopy over electron microscopy?

Answer 10

• light microscopes can visualise both living + non-living specimens
• light microscopy is relatively quick
• light microscopy is much less expensive

Question 11

What is magnification?

Answer 11

The size of the image compared to the real size of the object

Question 12

What is resolution?

Answer 12

The ability to tell the difference between two points

Question 13

Formula for magnification

Answer 13

Magnification = image size/actual size

Question 14

What is cell fractionation?

Answer 14

The separation of different organelles from each other by weight

Question 15

What are the 3 steps to cell fractionation?

Answer 15

Homogenisation, filtration and centrifugation

Question 16

What is the process of homogenisation?

Answer 16

• cells are put in a homogeniser to break u the plasma membrane of the cells releasing all the organelles into solution
• must be cold, isotonic and buffered

Question 17

Why does the homogenate need to be cold?

Answer 17

To reduce enzyme activity

Question 18

Why does the homogenate need to be buffered?

Answer 18

Because drastic changes in pH can harm organelles

Question 19

Why does the homogenate need to be isotonic?

Answer 19

So that water doesn’t move in or out of cells

Question 20

What happens in filtration?

Answer 20

The cell lysate needs to be filtered through a gauze which removes non-homogenised cells, cell debris and tissue debris

Question 21

What happens in centrifugation?

Answer 21

• the lysate solution is placed into a tube which is centrifuged at a low speed
• the heaviest organelles (nuclei) settle at the bottom as a pellet
• remaining organelles stay in supernatant which is put into another tube
• tube is then ultra centrifuged and again a pellet and supernatant is formed
• process is repeated until all the organelles have been separated out

Question 22

Order of fractionation

Answer 22

Nucleus
Chloroplast
Mitochondria
Lysosomes
Endoplasmic reticulum
Ribosomes