Microscopes And cell fractionation Flashcards
What is resolution?
Resolution is how detailed the image is.
It is how well a microscope distinguishes between two points that are close together.
If a microscope cannot separate two objects, increasing the magnification won’t help.
What is magnification?
Magnification is how much bigger the image is than the specimen you are looking at.
How do you calculate magnification?
Magnification = length of image / length of specimen
How do you go from nanometers to millimetres?
/ 1,000,000
How do you go from micrometres to millimetres?
/ 1000
How do you go from millimetres to nanometres?
X 1,000,000
How do you go from micrometres to nanometres?
X 1000
How do you go from millimetres to micrometres?
X 1000
Put these in orders from smallest measurement to largest:
Micrometres, nanometres, millimetres
Nanometres
Micrometres
Millimetres
What is the unit of micrometres?
um
What is the unit of nanometres?
Nm
What is the maximum resolution of light microscopes?
0.2 micrometres (Um)
What is the maximum magnification of light microscopes?
X 1500
Why do electron microscopes give a more detailed image than light microscopes?
They have a greater resolution.
What is the maximum resolution of electron microscopes?
0.0001 micrometres (Um)
What is the maximum magnification of electron microscopes?
X 1,500,000
Why does electron microscopes have higher magnifications (and resolutions?) than light microscopes?
The wavelength of an electron is a lot shorter than light waves
Disadvantages of electron microscopes?
Produce black and white images (can be coloured using computers)
Sometimes contain artefacts (dust and scratches) from preparing the specimen, affecting the clarity of the image
How do transmission electron microscopes work?
They use electromagnets to focus a beam of electrons that are transmitted through the specimen. Denser parts of the specimen absorb more electrons, making them look darker on the image.
How do scanning electron microscopes work?
They scan a beam of electrons across the specimen. This knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image. The image shows the surface of the specimen and they can be 3D.
What are the advantages of TEMs?
Higher resolution images (than SEMs) so shows small objects
What are the disadvantages of TEMs?
Can only be used on thin specimens
Can only be used in a vacuum so cannot be used on living organisms
What are the advantages of SEMs?
Can be used on thick specimens
Can be 3D
What are the disadvantages of SEMs?
Give lower resolution images (than TEMs)
Can only be used in a vacuum and so cannot be used on living organisms.
Why can electron microscopes only be used in a vacuum?
Something I imagine to with other atoms affecting the electrons
What is cell fractionation?
The process of looking at the organelles in a cell
What are the three stages of cell fractionation?
Homogenisation
Filtration
Ultracentrifugation
Describe homogenisation
Breaking up the cells
Vibrate of grind the cells
This breaks the plasma membrane, releasing the organelles into a solution
This must be kept ice cold to reduce the activity of enzymes that break up the organelles
This must be isotonic to prevent cells bursting or shrivelling due to osmosis
Describe filtration
Solution of organelles filtered through a gauze, getting rid of the tissue debris (connective tissue). Organelles are smaller so they pass through
Describe unltracentrifugation
This is the process of separating out organelles
The cell fragments are poured into a tube, this is put into a centrifuge (a machine that separates material by spinning). It is spun at a low speed. Heaviest organelles flung to the bottom, forming a sediment. The rest are suspended in supernatant. Supernatant is drained off into another tube and spun again at a higher speed, this process is repeated until all organelles are separated out.
What is the weight of organelles from largest to lowest?
Nuclei Mitochondria Lysosomes Endoplasmic reticulum Ribosomes
What would you expect to find in the pellet two formed from the centrifuge?
Mitochondria
What would you expect to find in the pellet one formed from the centrifuge?
Nuclei
What would you expect to find in the pellet three formed from the centrifuge?
Lysosomes
What would you expect to find in the supernatant three formed from the centrifuge?
Endoplasmic reticulum / ribosomes
