Microscopy

Question 1

What are the 3 types of microscope?

Answer 1

• optical (light) microscope
• transmission electron microscope (TEM)
• scanning electron microscope (SEM)

Question 2

How does an optical (light) microscope work?

Answer 2

• beam of light is condensed to create an image

Question 3

What are the limitations + advantages of optical microscopes?

Answer 3

• poorer resolution bc light has a longer wavelength so smaller organelles in cell aren’t visible
• lower magnification
• colour image
• can view living samples

Question 4

How does a transmission electron microscope (TEM) work?

Answer 4

• beam of e-s is condensed (by electromagnets) to create image
• e- gun produced beam of e-s, which pass through specimen
• denser parts absorb more e-s, so appear darker
• produces a 2D image

Question 5

What are the limitations + advantages of transmission electron microscopes?

Answer 5

• sample must be non-living, bc must be in a vacuum as e-s are absorbed by air
• black + white images bc samples must be stained
• sample must be thin
• higher resolution bc e- have a short wavelength
• higher magnification

Question 6

How does a scanning electron microscope work?

Answer 6

• beam of e-s is condensed (by electromagnets) to create image
• e-s are beamed onto surface + are scattered in diff ways, depending on contours
• produces a 3D image

Question 7

What are the limitations + advantages of scanning electron microscopes?

Answer 7

• sample must be non-living, bc must be in a vacuum as e-s are absorbed by air
• black + white images bc samples must be stained
• higher resolution bc e- have a short wavelength
• higher magnification
• specimen don’t need to be thin

Question 8

What is the difference between magnification + resolution?

Answer 8

• magnification: how many times larger image is compared to object
• resolution: minimum distance between 2 objects in which they can still be viewed as separate

Question 9

What formula can be used to measure structures viewed under an optical microscope?

Answer 9

• image = actual x magnification (I = AM)

Question 10

How do you convert units from m - mm - μm - nm?

Answer 10

• m - mm = x1000
• mm - μm = x1000
• μm - nm = x1000

Question 11

What is cell fractionation used for?

Answer 11

• to isolate diff organelles, so their structure + function can be studied

Question 12

What are the 2 stages of cell fractionation?

Answer 12

• homogenisation
• ultracentrifugation

Question 13

Describe homogenisation in cell fractionation.

Answer 13

• plasma membrane of cells are broken up using a blender, in a cold, isotonic, buffered solution, to release organelles into solution
• solution is then filtered to remove large cell debris

Question 14

Why are cells broken up in a cold, isotonic, buffered solution?

Answer 14

• cold: to dec enzyme activity, so organelles aren’t damaged by enzymes released when cells break open
• isotonic: so water potential is same, preventing osmosis, so organelles don’t shrivel or burst
• buffered: solution has a pH buffer, to prevent organelles, proteins + enzymes from denaturing

Question 15

Describe ultracentrifugation in cell fractionation.

Answer 15

• filtered solution is spun at a low speed in a centrifuge
• centrifuge force separates organelles into densities, + so forms a pellet containing largest + heaviest organelles
• supernatant is removed + spun at increasingly higher speed, forming a pellet w a lighter organelle
• process repeats