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
