Microscopy Flashcards
1
Q
What is the difference between resolution and magnification?
A
- Resolution is the ability to distinguish between 2 points -> if you cannot resolve 2 points, you see them as one
- Increasing the size of an image but does not increase the resolution
- The object will appear larger but just as blurred
2
Q
What are the types of microscope ?
A
- Optic microscope
- Transmission electron microscope
- Scanning electron microscope
3
Q
How do light microscopes work?
A
- Specimens are illuminated with light
- The light is focused using glass lenses and viewed using the eye. All light microscopes today are compound microscopes, which means they use several lenses to obtain high magnification.
4
Q
What are the advantages and disadvantages of light microscopes?
A
- Poor resolution because of relatively long wavelength of light. The resolution is 200nm so anything closer then that will only be seen as one point.
- Only 2D slice can be observed
- Specimens can be living or dead
- You can see colour
5
Q
How do TEM microscopes work?
A
- Electron beam passes through a thin section of a specimen,
- Parts of the specimen absorb electrons and therefore appear dark, other parts of the specimen allow the electrons to pass through and so appear bright.
6
Q
What are the advantages and disadvantages of using TEM ?
A
- Specimens must be thin
- Specimens must be dead as they must be in a vacuum so that the electrons can pass through.
- An image that can be photographed
produced on a screen - Black and white
- 2D slice
- Really high resolution because of the shorter wavelength of electrons (0.1nnm)
- Can magnify up to 500,000 times
7
Q
How does SEM work?
A
- SEM directs a beam of electrons on the surface of the specimen and the beam is passed back and fourth across the specimen. The electrons are scattered and detected.
8
Q
What are the advantages and disadvantages of SEM?
A
- All the limitations of the TEM also apply to the SEM except that the specimen doesn’t need to be really thin
- 3D image can be generated. Allows detailed study of surfaces.
- Lower resolving power than TEM
- Resolving power of around 20nm
9
Q
Equation for magnification
A
Magnification = image size / actual size
10
Q
Compare the units used in microscopy to a meter
A
- Millimeter (mm) -> 1/1000
- Micrometer (um) -> 1/1,000,000
- Nanometer (nm) -> 1/1,000,000,000
- Picometer (pm) -> 1/1,000,000,000,000
11
Q
How to measure the actual size of a specimen using a microscope ?
A
- Use an eyepiece graticule and stage micrometer
- Before you can use the graticule it must be calibrated.
- To do this you line up the scale on the eyepiece with that of the micrometer using the objective lense .
- For example, you may use the 400x objective lens that magnifies 400 times.
- Suppose this shows that 50 graticule units are equivalent to 10 micrometer units.
- If each micrometer unit is 10um then each micrometer unit equals 2 um.
- If an objective lens magnifying 100 times is then used, each graticule unit would be equivalent to 8um.
12
Q
Rules for drawing low power drawings
A
- Identify the different tissue, using high power to help if necessary
- Draw all tissue and completely enclose each tissue by lines
- Don’t draw individual cells
- Accuracy is important- the specimen will not necessarily look like a textbook drawing
- A representative portion may be drawn if the structure if the structure is symmetrical
13
Q
Rules for drawing high power drawings
A
- Draw only a few representative adjacent cells. If all the cells are similar, then three cells is often sufficient to show cell structure and how cells are arranged in relation to each other
- Don’t shade
14
Q
Steps of differential centrifugation
A
- Tissue is cut up and kept in a cold, isotonic buffered solution
- Cut up tissue further broken down in a homogeniser
- Homogenised tissue is spun in an ultracentrifuge at a low speed for 10 minutes
- Filtrate spun at different speeds to form different pellets
15
Q
Explain the first step in differential centrifugation
A
- Cold -> reduce enzyme activity so organelles aren’t digested
- Isotonic -> prevent organelles bursting or shrinking as a result of the osmotic gain or loss of water
- Buffered -> maintain constant PH
