Name the 3 types of microscope
Optical (light) microscope
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
What is the resolution of an optical microscope determined by?
The wavelength of light
What is the resolution of an electron microscope determined by?
The wavelength of the beam of electrons
What is the technical name for a microscope image
A micrograph
What does a scale bar show?
The size of actual objects in an image relative to the scale
Compare optical to electron microscopes in terms of:
1) how the image is formed
2) resolution
3) magnification
4) whether images produced are in colour or not
5) whether living samples can be viewed
1) optical - beam of light condensed to form an image. Electron - beam of electrons condensed to form an image
2) optical - worse, electron - better
3) optical - lower, electron - higher
4) optical - colour, electron - black and white
5) optical - can view living samples, electron cannot
Why is the resolution of optical microscopes worse than for electron microscopes?
Light has a longer wavelength than the wavelength of electrons
Why are images formed by electron microscopes black and white?
The sample must be stained before viewing
Why can’t electron microscopes be used to view living samples
Sample must be in a vacuum as electrons are absorbed by air
How does a transmission electron microscope work?
Extremely thin specimens stained and placed in a vacuum. An electron gun produces a beam of electrons that pass through the specimen. Some parts absorb the electrons and appear darker.
What can TEMs be used to view?
The internal structure of cells and inside of organelles (unlike SEM)!
What type of images are formed by TEMs and SEMs?
TEM - 2D
SEM - 3D
How does a scanning electron microscope work?
Electrons are beamed onto the surface and they’re scattered in different ways depending on the contours.
Why don’t specimens in SEMs need to be thin?
The electrons aren’t transmitting through the specimen, unlike TEMs
What type of microscope has an eyepiece graticule?
An optical microscope (electron microscopes don’t have them!)
What piece of equipment is used to calibrate the eyepiece graticule?
Stage micrometer
What is a stage micrometer?
A glass slide with a scale on it which you place on the stage
Why must the eyepiece graticule be recalibrated every time you change the objective lens (and therefore magnification) used?
It’s used to measure the size of objects viewed under the microscope and the distance represented by the space between each division in an eyepiece graticule changes as magnification does
State the method for calibrating the eyepiece graticule
1) align the zero mark on the stage micrometer with the zero on the eyepiece graticule scale while looking through the eyepiece.
2) count how many divisions on the eyepiece graticule scale fit into a certain number of divisions on the micrometer scale
3) length of each division on the stage micrometer (100nm for us) can be multiplied by the number of divisions on it (to find total length of stage micrometer)
4) divide that value by the number of eyepiece graticule divisions to calculate what 1 division is worth at that magnification
5) repeat for different magnifications
6) the scale can now be used to determine the size of the sample viewed
Why is HCl added to the tissue sample?
To fix/ kill the cells so they remain in their stage of mitosis, so that the cellulose cell wall becomes more porous which allows uptake of toluidene blue stain
Why is a stain added to the tissue sample?
Toluidene stains the chromosomes blue so that they can be viewed under the light microscope
Why is a mounted needle used?
To lower the cover slip at an angle to prevent air bubbles under the cover slip
Why is only the first 2mm of root tip used?
The tip is the growing region, therefore the cells should be actively dividing (mitosis should be occurring here).
Why press down on the cover slip?
To squash the sample to get a thin layer of cells that light can pass through so different stages of mitosis can be clearly viewed
