2.2 Microscopy Flashcards
Name the 3 types of microscope?
Optical, Electron (Transmission Electron Microscope, Scanning Electron Microscope)
Magnification Formula:
magnification = size of image / size of real object
Magnification is …
how many times bigger the image produced by the microscope is than the real-life object you are viewing
Resolution is …
the ability to distinguish between objects that are close together
Optical microscopes have a … (1) resolution, because visible light has … (2)
(1) low
(2) long wavelengths
How does an optical microscope produce an image?
Uses convex lenses to magnify images of small samples with visible light.
Advantages of Transmission Electron Microscope
- very high resolution images
- allows the internal structures within cells to be seen
How does an electron microscope produce an image?
Uses beams of electrons to form an image.
TEMS: Use electromagnets to focus a beam of electrons on the sample, this beam of electrons is transmitted through the specimen. Denser parts of the specimen absorb more electrons making these parts appear darker on the final image produced.
SEMS: Scan a beam of electrons across the specimen. This beam bounces off the surface of the specimen and the electrons are detected, forming an image.
Disadvantages of Transmission Electron Micrscope
- can only be used with very thin specimens
- can not observe live specimens
- lengthy treatment required to prepare specimens means that artefacts can be introduced
- do not produce a colour image
Advantages of Scanning Electron Microscope
- can be used on thick specimen
- allow the external, 3D structure of specimens to be observed
Disadvantages of Scanning Electron Microscope
- lower resolution than TEM
- can not observe live specimens
- do not produce a colour image
Optical vs Electron (size)
Optical: Small and portable
Electron: Large and installation means it can’t be moved
Optical vs Electron (magnification + resolution)
Optical: mag → x2000 res → 200nm
Electron: mag → >x500,000 res → 0.5nm (larger and higher)
Optical vs Electron (specimens)
Optical: Live
Electron: Dead
Optical vs Electron (preparation)
Optical: Simple
Electron: Complicated
Optical vs Electron (vacuum)
Optical: ✗
Electron: ✓
What is cell fractionation?
Separating cell organelles from each other.
What are the steps of cell fractionation?
- Homogenisation
- Filtration
- Ultracentrifugation
What is homogenisation?
The biological term for the breaking up of cells.
The homogenate solution must be …
Ice Cold
Buffered
Isotonic
Ice Cold?
To reduce the activity of enzymes that break down organelles.
Buffered?
To prevent organelle proteins from denaturing.
Isotonic?
So water potential is not affected to prevent water moving into organelles by osmosis which could lead to cell damage.
What does the homogeniser do?
This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate.
Why is the homogenate filtered?
To remove any large cell debris that were not broken up.
What is ultracentrifugation?
The filtrate is placed into a tube and the tube is placed in a centrifuge.
Then the filtrate is spun at increasing speeds to separate the different organelles.
Order of mass of the organelles.
- Nuclei
- Chloroplasts (for plant tissues)
- Mitochondria
- Lysosomes
- Endoplasmic reticulum
- Ribosomes
In what order are the organelles separated.
Heaviest organelles (nucleus/mitochondria) first at the slowest speeds. Lighter organelles last at faster speeds.
Process of ultracentrifugation.
Spinning causes the largest, heaviest organelles 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, known as the supernatant.
This supernatant is re-spun at a higher speed.
Process of ultracentrifugation.
Spinning causes the largest, heaviest organelles 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, known as the supernatant.
This supernatant is re-spun at a higher speed.
