Investigating thestructure of cells

• Instruments that magnify the image of an object.
• Light microscopes have long wavelength of light rays.
• This means that a light microscope can only distinguish between two objects if they are 0.2 micrometers or more apart.
• better microscopes used a beam of electrons.
• Beams of electrons have a shorter wavelength than light.
• They can distinguish two objects as close as 0.1nm.

• Material under the microscope is the object.
• The appearance if the material when viewed under a microscope is reffered to as the image.
• magnification= size of image/ size of object
• When calculating this the measurements of the image and the object must be in the same units, e.g. cm.
• size of object= size of image/ magnification.
• 1m-1m
• 1cm= 1/100m
• 1mm=1/1,000m
• 1um=1/1,000,000m
• 1nm=1/1,000,000,000m

• Resolution or resolving power of a microscope is the minimum distance apart that two objects can be in order for them to appear as seperate objects.
• Resolving power depends on the wavelength or form of radiation used.
• Light microscope- 0.2 um
• Greater resolution means greater clarity.
• The image produced is clearer and more precise.
• Increasing the magnification will increase the size of the image, but does not always increase the resolution.
• Every microscope has a limit of resolution.
• Up to this point increasing the magnification will reveal more detail but beyond this point  increasing the magnification will not do this.
• The object, while appearing larger, will just be more blurred.

• In order to study the structure and function of various organelles it is necessary to obtain large numbers of isolated organelles.
• Cell fractionation is the process where cells are broken up and different organelles they contain are seperated out.

Before the process can begin, the tissue is placed in a cold, isotonic, buffered solution.

Cold - to reduce enzyme activity that might break down the organelles.

Isotonic - to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water. It is a solution that has the same water potential as the original tissue.

Buffered - to maintain a constant pH

• Cells are broken up by a homogeniser (blender)
• This releases the organelles from the cell
• The resultant fluid - the homogenate- is filtered.
• This removes any whole cells or large pieces of debris

• The process by which the fragments 0in the homgenate are seperated. This is in a machine called an untracentrifuge. This spins the homogenate at high speeds in order to create a centrifugal force.
• The process in animals is:
• 1) The tube of filtrate is placed in the centrifuge and spun at a low speed.
• 2)The heaviest organelles, the nuclei, are forced to the bottomof the tube where they form a thin sediment of pellet.
• 3)The supernatant is removed, leaving the sediment of nuclei.
• 4)The supernatant is transferred to another tube and spun in the ultracentrifuge t a higher speed than before.
• 5)The next heaviest organelles, the mitochondria, are forced to the bottom of the tube.
• 6)The process is continued in this way so that, at each increase in speed, the next heaviest organelle is sedimented and seperated out.
• The order is: - Nuclei, - Mitochondria, - Lysosomes, - Ribosomes.