Methods of studying cells

What is the cell theory?

All living things are made up of cells, cells are the basic units of structure and function in living things, new cells are produced from existing cells

What is the basic unit to measure cells?

Micrometer

How many nm in a micrometer?

1000 nm in a micrometer

How many micrometers in a mm?

1000 in 1 mm

What are te 3 types of microscopes?

Optical, electron microscopes: transmission and scanning

What is magnification?

How many times larger the image is compared to the object

What is resolution?

The minimum distance between 2 objects in which they can still be viewed as separate

Resolution in light microscopes and electron microscopes

Light microscopes resolution depends on the wavelength of light, in electron microscopes it depends on the wavelength of electrons

Features of light microscopes

• beam of light condensed to create the image

• poor resolution as light has a longer wavelength

• lower magnification

• colour images produced

• can view living samples

Features of electron microscopes

• electromagnets condense beam of electrons to create image

• higher resolving power as electrons have a shorter wavelength

• higher magnification

• B+W images

• sample in a vacuum so is dead (electrons would have been absorbed by air)

What is a transmission electron microscope (TEM)?

Extremely thin specimens stained and placed in a vacuum, electron gun produces a beam of electrons which pass through sample, some parts of specimens absorb electrons and appear dark.

Evaluate TEM

• 2D image produced

• shows detailed images of internal cell structure

• image may contain artefacts

What is a scanning electron microscope (SEM)?

Electrons beamed onto surface and scattered in different ways depending on contours

Evaluate SEM

• produces a 3D image

• specimens don't need to be as thin as electrons are not transmitting through

• lower resolving power than TEM

• image may contain artefacts

What is an eyepiece graticule?

Scale on glass disc inside eyepiece of optical microscopes used to measure size of objects being viewed under microscope

How is an eyepiece graticule calibrated?

• line up the stage micrometer (slide w a scale on it) and eyepiece graticule whilst looking through the eye piece

• count how many divisions on the eye piece graticule fit into one division on the micrometer scale, each division on the micrometer is 10 micrometer- this can be used to calculate what one division on the eye piece graticule is at the current magnification

What is cell fractionation?

The process where cells are broken up and the different organelles they contain are separated out, so individual organelle structures and functions to be studied

What must the cells be prepared in before cell fractionation?

A cold, isotonic and buffered solution

Why is the solution cold?

reduce enzyme activity that may break down the organelles

Why is the solution isotonic?

Same water potential as tissue to prevent osmosis as it could cause the organelles to shrivel or burst

Why is the solution buffered?

So that the pH does not fluctuate. Any change in pH could alter the structure of the organelles or affect the functioning of enzymes

Stage 1 of cell fractionation

HOMOGENISATION- cells are broken up by a homogeniser (blender)which releases the organelles. The resultant fluid (homogenate) is then filtered to remove large cell debris

Stage 2 of cell fractionation

ULTRACENTRIFUGATION- fragments in filtered homogenate are separated in a machine called a centrifuge, organelles separated according to densities. Done by differential centrifugation.

Ho does differential centrifugation work?

Centrifuge spins at a low seed first and centrifugal forces causes pellets of the most dense organelles to form at the botttom. Supernatant (liquid) is removed leaving behind a pellet of organelles. Supernatant is transferred to another tube and spun at a faster speed than before to remove next pellet or organelles. Process continued so that at each increase in sped the next heaviest organelle is sedimented and separated

What order of density are organelles separated?

Nuclei, chloroplasts (plant only), mitochondria, lysosomes, E.R, ribosomes