- 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.
- 1cm= 1/100m
- 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
Step 1) Homogenation
- 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
Step 2) Ultracentrifugation
- 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.