Cells: Methods of studying cells Flashcards
What is resolution
- The ability to distinguish two very small adjacent structures as separate (the higher the resolution the better the clarity and detail of the image)
State the two types of microscopes
- Optical microscopes
- Electron microscopes
State the two types of electron microscope
- Transmission electron microscope
- Scanning electron microscope
Outline the features of transmission electron microscopes
- A beam of electrons is transmitted through the specimen with electromagnets. Denser parts of the specimen absorb more electrons, thus appearing darker in the image.
- Higher resolution than SEM
- Specimens must be thin
- Complex staining method required and image cannot be coloured
Must be in a vacuum, so live specimens can’t be observed
Outline the features of scanning electron microscopes
- A beam of electrons is scanned across the specimen which knocks electrons off the specimen. These electrons are gathered in the cathode ray tube to form an image.
- Lower resolution than TEM.
- Specimens can be thick because SEM doesn’t penetrate
- Image can be coloured
- Can produce a 3D image, but cant see inside the specimen
- Must be in a vacuum, so live specimens can’t be observed
State the advantages of using an electron microscope over an optical microscope
State the advantages of using an scanning electron microscope
- They can be used on thick or 3D specimens
State the disadvantages of using an scanning electron microscope
- They have a lower resolution image than transmission electron microscopes
- They do not produce an image colour (unlike optical microscopes)
- They cannot be used to observe live specimens
State the advantages of using an transmission electron microscope
- They give high-resolution (higher than SEM) - allowing internal structures within cells to be seen
State the disadvantages of using an transmission electron microscope
- They do not produce an image colour (unlike optical microscopes)
- They cannot be used to observe live specimens
- They can only be used with thin specimens
Briefly outline the three stages of cell fractionation
- Homogenisation - Breaking up the cells
- Filtration - Getting rid of the big bits
- Ultracentrifugation - Separating the organelles
Describe the procedure of homogenisation
- Place a sample of tissue (containing the cells needing to be broken up) into a cold, isotonic buffer solution
- Put it into a homogeniser which grinds up the cells - breaking the plasma membrane and releasing the organelles into the solution (called the homogenate)
Describe the procedure of filtration
- The homogenate is filtered through a gauze, separating out large cell debris
- This leaves a solution containing a mixture of organelles (called the filtrate)
State what conditions the extraction buffer in homogenisation need to be in and why
- It has to be ice cold - to stop degradation by enzymes
- It has to have a balanced pH - so the structure of the cell doesn’t change
- Buffer has to be isotonic - so the organelles don’t burst or shrivel up
Describe the procedure of ultracentrifugation
- Filtrate is placed into a tube and the tube is placed in a centrifuge
- The filtrate is first spun at a low speed - causing the largest, heaviest organelles (such as the nuclei) to settle at the bottom of the tube, where they form a thick sediment known as a pellet
- The rest of the organelles stay suspended in the solution above the pellet
- The supernatant is drained off and placed into another tube, which is spun at a higher speed
- Once again, this causes the heavier organelles (such as the mitochondria) to settle at the bottom of the tube, forming a new pellet and leaving a new supernatant
- This process is repeated - increasing the speed every time the supernatant is drained into a different tube until all the organelles have been separated
What are the two main types of studying cells
- Magnification
- Cell fractionation
What is magnification
The process of enlarging an object in appearance
What is image size
It is how big the object appears to be in a picture or drawing
What is the actual size
The real size of an object
What is the equation for magnification
Magnification = image size / actual size
State what cell fractionation is
It is the process the used to separate cellular components while preserving individual functions of each component
Order of organelles by mass (Biggest to smallest)
- Nucleus
- Chloroplasts
- Mitochondria
- Lysosomes
- Endoplasmic reticulum
- Ribosomes
State the maximum resolution of an optical microscope
0.2 µm
State the maximum resolution of an electron microscope
0.1 nm
State the maximum magnification of an optical microscope
x1500
State the maximum magnification of an electron microscope
x1,500,000
What is a centrifuge
A machine that separates materials by spinning
What is a supernatant
Liquid suspension of lighter organelles (lighter density) which forms above the pellet
What is a pellet
Liquid suspension with the heaviest organelles settling at the bottom of the tube forming a thick sediment
Every time a supernatant is drained of into another tube during ultracentrifugation it is spun faster, why
The higher the speed the smaller the organelle you are going to obtain
What’s the difference between how scanning electron microscopes and transmission electron microscopes function
- SEM creates an image by detecting reflected or knocked-off electrons
- TEM uses transmitted electrons (electrons that are passing through the sample) to create an image.
Why do light microscopes have a lower resolution than an electron microscope
Becuase it has a shorter wavelength
