Microscopes and Cell Fractionation

Question 1

Magnification

Answer 1

Magnification of an object is how many times bigger the image is when compared to the object. Use the formula magnification= Image size/actual size

Question 2

Resolution

Answer 2

Resolution is defined as how well you can distinguish between two points. Greater resolution means greater clarity, so the image produced is clearer and more precise.

Question 3

How do Light Microscopes Work?

Answer 3

Light from the mirror is reflected up through the specimen (object) into the powerful objective lens which produces the first magnification. Image produced by the lens is then magnified again by the eyepiece lens

Question 4

Light Microscopes Disadvantages

Answer 4

Long wavelength of light means light microscopes have a poor resolution. Can only distinguish between 2 objects if they are 0.2μm away.
Low magnification (Max magnification- x 1500)
Can’t see Mitochondria, Endoplasmic Reticulum, Golgi apparatus and Ribosomes

Question 4

Light Microscopes Advantages

Answer 4

-Easy to use
-Cheap to purchase
-Shows true colours of the specimen
-Can view live specimens

Question 5

How does a Transmission Electron Microscope Work?

Answer 5

A beam of electrons is focused on the thin specimen. Some parts of the specimen absorb the electrons thus appear darker and other parts allow the electrons to pass through so appear bright. The image produced on the screen is photographed to produce a photomicrograph.

Question 6

Disadvantages of TEM

Answer 6

-May contain artefacts which distorts the real image
-Whole system is carried in a vacuum (so electrons don’t interact with air molecules) but this means live specimens can’t be viewed
-Complex staining process
-Doesn’t show true colours of the specimen
- Very expensive

Question 7

Advantages of TEM

Answer 7

High resolution
High Magnification

Question 8

How does a Scanning Electron Microscope work?

Answer 8

A beam of electrons is directed onto the surface of a thin specimen. The beam passes back and forth across a section of a specimen in a regular pattern. The electrons are then spread based on the outlines of the specimen surface. A 3D image is created based on the computer analysis of the pattern of scattered electrons.

Question 9

Disadvantages of SEM

Answer 9

-Very expensive
-Sample needs to be extremely thin
-Shows false colour
-Extensive training required to use an SEM
-Live specimens can’t be viewed

Question 10

Advantages of SEM

Answer 10

High resolution (Max- 20 nm)
High magnification (Max- x 100000)

Question 11

Cell Fractionation: Why Cold?

Answer 11

To reduce/ prevent enzyme activity that might break down the organelle

Question 12

Cell Fractionation: Why Buffered?

Answer 12

Maintains the same PH as a change to the PH could alter the structure and function of the organelle

Question 12

Cell Fractionation: Why Isotonic?

Answer 12

Prevents organelles from bursting or shrinking due to an osmotic gain or loss of water

Question 13

First Stage of Cell Fractionation

Answer 13

Homogenisation - Cells are broken up by a homegenisor (blender) releasing organelles from the cell. The homogenate is then filtered to remove any cells/large pieces of debris

Question 14

Final Stage of Cell Fractionation

Answer 14

Ultracentrifugation
Fragments in the filtered homogenate are separated using a centrifuge. The heaviest organelle falls to the bottom and forms a pellet the rest remains as a fluid at the top called supernatant. The supernatant is removed and placed in another tube and spun at a higher speed. The next heaviest organelle is forced to the bottom.

Question 15

Order of Density of Organelles? Most to Least

Answer 15

Nuclei, Mitochondria, Chloroplasts, Endoplasmic Reticulum, Golgi apparatus and Ribosomes.