microscopes and cell fractionation and ultracentrification

Define magnification and resolution

Magnification-how many times larger the image is compared to the object

Resolution-minimum distance between two objects in which they can still be viewed as seperate

Optical (light) microscope and electron microscope (scanning or transmission)

Light - beam of light is condensed to create the image

• poorer resolution due to light having a longer wavelength (small organelles are not visible)

• Lower magnification

• Colour image

• Can view living samples

Electron - beam of electrons is condensed to create image

• higher resolving power as electrons have a short wavelength

• Higher magnification

• Back and white images

• Sample must be in vacuum so has to be non-living

Electron microscope and transmission electron microscopes and scanning electron microscope

EM - electrons are absorbed by air, which is why samples must be in a vacuum, image is black and white as samples have to be stained

TEM - extremely thin specimens are stained and placed in a vacuum, an electron gun produces a beam of electrons that pass through the specimen, some parts absorb the electrons and appear dar, image produced is 2D and shows detailed images on internal structure of cells

SEM - specimens do not need to be thin, as the electrons are not transmitting through, instead the electrons are beamed onto the surface and the electrons are scattered in different ways depending on the contours, produces a 3D image

How to work out magnification

Image size = actual size x magnification

Cell fractionation

Cells are broken open to release contents and organelles are then separated

Cells must be prepared in a cold, isotonic and buffered solution:

• Cold: to reduce enzyme activity, when cell breaks open enzymes are released which could damage the organelles

• Isotonic: must be the same water potential to prevent osmosis as this could cause the organelles to burst

• Buffered: the solution has a pH buffer to prevent damage to organelles

Steps of cell fractionation

1. Homogenisation: break open the cell (homogenised) using a blender in a cold, isotonic and buffered solution, solution is filtered to remove large cell debris

2. Ultracentrifugation: filtered solution is spun at different speeds in a centrifuge, organelles seperate according to their densities

3. This is done through differential centrifugation: centrifuge spins and centrigufal forces cause pellets of the most dense organelles to form at the bottom, centrifuge is first spun at low speed and the process is repeated ta increasingly faster speeds, each time the supernatent is removed and leaved behind a pellet of organelles, supernatent is spun again to remove next pellet of organelles

4. Nuclei, chloroplast, mitochondria (if plant cells), lysosomes, endoplasmic rectilium, ribosomes