Lesson 2 - Microscopy Flashcards
Magnification definition:
How many times larger the image is than the actual size of the object being viewed.
How to see details?
Magnification does not change the visibility (details) of the image. Resolution is needed.
Resolution definition:
Minimum distance between two objects where they can still be seen as two separate objects
What limits resolution?
Diffraction
Diffraction definition:
The tendency of light waves to spread as they pass close to physical structures such as those present in specimens being studied. The structures present in specimens are very close to each other and the light reflected from individual structures can overlap due to diffraction. This means that structures are no longer seen as separate entities and detail is lost.
Optical microscopy structures:
If structures are closer than half a wavelength of light, then structures can not be seen individually.
How to increase resolution?
Electron microscopy
Use beams of electrons, which have a wavelength a thousand times shorter than light.
- Electron beams are still diffracted but the shorter wavelength means that beams can be much closer before they overlap.
- This means that structures which are smaller and much closer together can be seen separately without diffraction blurring the image.
Magnification equation?
Magnification = Size of Image / Actual size of object
Limiting factor of light microscopy?
Resolution
Electron microscopy?
- A beam of electrons with a wavelength of less than 1mm is used to illuminate the specimen.
- More detail of cell ultra structure can be seen because electrons have a much smaller wavelength than light.
- They produce images with a magnification of up to x 500,000 and still have clear resolution.
Disadvantages of electron microscopy?
- Expensive pieces of equipment
- Can only be used in a carefully controlled environment in a dedicated space.
- Cells can be damaged by the electron beams
- The preparation process is complicated and there is the complication of artefacts (structures that are created in the preparation process). As techniques improve they can be eliminated.
The two types of electron microscopes?
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
Transmission Electron Microscope (TEM):
- Beam of electrons is transmitted through a specimen and focused to produce an image.
- This is similar to light microscopy
- Best resolution (with resolving power) of 0.5 nm
Scanning electron microscope (SEM):
- Beam of electrons sent across the surface of a specimen and reflected electrons are collected.
- Resolving power is from 3 - 10 nm
- Resolution is not as good as transmission electron microscope
- 3D images created, giving valuable information about the appearance of structures.
Preparation for electron microscopy?
- Fixation using chemicals or freezing
- Staining with heavy metals
- Dehydration using solvents
TEM: Set in resin and may be stained again
SEM: Fractured to expose the inside. Then stained with heavy metals again.
Why are specimens prepared specifically for electron microscopes?
The inside of the microscope is a vacuum.
Resolving power of light vs electron microscopes?
Light: 200 nm
Electron: 0.5 nm / 3-10 nm
Example of artefact in light microscopy?
Air bubbles under cover slip
What was the name given to the artefact thought to be an inward fold of the cell membrane in prokaryotic cells?
Meosome
What did scientists believe the meosome was for?
Surface area of the folded surface was believed to be an important site of process of oxidative phosphorylation.
How was the meosome disproved?
A non-chemical technique was developed called cryofixation. Mesosomes were no longer visible.
Wavelength of light:
400 - 700 nm
What do old light microscopes and modern light microscopes have in common?
- Stage
- Eyepiece lens
- Focusing dial
What is the use of an electromagnet in electron microscopes?
Focus the beam of negatively charged electrons
What produced the beam of electrons?
Electron gun
