Studying cells

what do light microscopes use to work?

Uses beam of light

What does electron microscope use to work?

Uses beam of electron

What is the max magnification of a light microscope?

✖1,500

What is the max magnification of a electron microscope?

✖1,500,000

What is the max resolution of a light microscope?

0.2 µm

What is the max resolution with the electron microscope?

0.002 µm

Can you magnify live specimens using a light microscope?

Yes

Can you magnify live specimens with an electron microscope?

No

In electron microscope why can we not view a live specimen?

Specimens have to be kept in a vacuum as other air particles will absorb electrons and interfere with results (live specimens would die due to lack of air)

Light microscopes have lower resolution and magnification.

This means we cannot see what organelles?(3)

• ribosomes

• Lysosomes

• Endoplasmic reticulum

Why do electron microscopes have a better resolution than light microscopes?

Electrons have shorter wavelength than light

What are the two types of electron microscopes?

• transmission electron microscope(TEM)

• Scanning electron microscope(SEM)

How does a transmission electron microscope work?

A beam of electrons are transmitted through the specimen

On denser parts of the specimen what happens when using an TEM?

Denser parts of the specimen absorb more electrons = darker on the image

How does an scanning electron microscope work?

Scans a beam of electrons across the specimen

What happens to the electrons when using a SEM?

Electrons are knocked of the specimen and are collected in a cathode ray tube

What does TEM show?

Can show internal structure of organlles

What does SEM show?

Can only show surface of specimen

What has higher resolving power, TEM or SEM?

TEM has higher resolution

What type of thickness has to be used for TEM and SEM?

• TEM = can only be used on thin specimens

• SEM = can be used for thick specimens

Is the image produced in 2D/3D for TEM and SEM?

• TEM= 2D

• SEM= 3D

What is the formula for magnification?

Magnification = image size / actual size

What is the definition of resolution?

The minimum distance that two objects can be apart for them to appear as separate objects

What is the definition for magnification?

How many times the image is larger than the specimen

What is cell fractionation?

The process by which cells are broken up and their organelles are separated out

What is the first step of cell fractionation?

1. Homogenisation

   • breaks up cells using a homogeniser (blender) or by vibrating them

   • Plasma membranes breaks and the organelles are released into the solution the cells were kept in

What qualities should the solution have where cells are kept in for cell fractionation?+why(3)

• cold- reduces enzyme activity which may hydrolyse organelles

• Isotonic- solution must have the same water potential= prevents cells from bursting or shrinking due to osmosis

• Buffer- maintains pH. A fluctuating pH could damage organelles or effect functioning enzymes

What is the second step of cell fractionation?

2. Filtration

   • The result fluid from homogenisation process (homogenate) is filtered to remove unbroken cells or cell debris

What is the third step in cell fractionisation?

3. Ultracentrifugation

   • The process by which different organelles in the filtered solution are separated out in the centrifuge

How does ultracentrifugation work?(6)

1. Mixture poured into test tube.

2. The tube is put into a test centrifuge

3. This is then spun at a low speed

4. heaviest organelle (e.g nuclei) are forced to the bottom to form the pellet. The suspension above the pellet = supernatant

5. The supernatant is drained off, poured into another test tube, then spun at a higher speed = mitochondria now found in pellet

6. Supernatant is drained off again & process repeats, but each time the tube is spun at a higher speed, light and less dense organelles are forced to the bottom & found in the pellet

• The order in which organelles are found in the pellet depends on the speed of the centrifuge.

• The ____ and less ____ organelles are found in the pellet at _____ speeds

• ORDER: N___ ,_____, l____, ER, R____.

• the lighter and less dense organelles are found in the pellet at faster speeds

• Nuclei, mitochondria, lysosomes, ER, ribosome