Electron Microscopy Flashcards
Define resolution
the ability to distinguish between two very closely positioned objects
Why is resolution important?
because magnification alone does not make an image clearer, it just makes it bigger
resolution is required to make an image clear
What is the maximum resolution of a light microscope? What does this mean?
0.2 micrometers with the 100x objective lens/total 1000x magnification
There must be at least 0.2 micrometers between the two spots to be able to see them as separate objects with a light microscope
What is the formula for resolution? what does each variable stand for?
D = 0.61 lambda/ n sin alpha
D = the minimum distance between two distinguishable objects
lambda = wavelength of light used
n = refractive index of medium between specimen and objective lens
alpha = angular aperture (half angle) of the cone of light entering the objective lens from the specimen
Why does resolution increase so dramatically with electron microscopes?
the shorter the wavelength, the better the resolution
electron microscopes use electrons as an energy source instead of light
electrons have shorter wavelengths than light
Which has a higher resolving power: electron or light microscope?
electron
Describe electron microscopes
Use an electron beam to pass through the specimen
What are the two kinds of electron microscopy?
Transmission EM
Scanning EM
Describe transmission EM
Forms images as a 2D slice of an object by using electrons that are TRANSMITTED through a specimen
What is the range of resolution for TEM?
3-5 Å = 0.00003-0.00005 micrometers
Describe scanning EM
Forms 3D images of an object by using electrons that are BOUNCED OFF the surface of a specimen
What is the range of resolution for SEM?
5-10 nm
What size of specimen would you use in a SEM?
larger sized specimens
T or F: With both SEM and TEM, you can see the internal organelles of a specimen
FALSE.
Only with TEM could you see the internal organelles of a specimen because the electrons transmit through it
You could not see with the SEM because the electrons bounce off the surface of the specimen
Describe the structure of an EM and how an EM works?
Top of column contains a cathode which is heated to provide a source of electrons
a vacuum is created inside the EM in order to direct the electrons downwards and reduce scattering
The beam of electrons is focused by electromagnetic lenses in the wall of the column
the specimen is on a small metal grid that is inserted into a grid holder which is inserted into the column of the EM
What is the purpose of creating a vacuum in EM?
In order to direct the electrons downwards toward the specimen
without the vacuum, the electrons would scatter by collision with gas molecules
What does it mean if spots on the image produced by an EM of the specimen are darker than others?
Fewer electrons hit that spot because the specimen was denser there
Why will electrons not hit the denser parts of a specimen?
electrons may reflect and scatter after hitting specimen
the greater number of specimen atoms, the greater the probability of electrons scattering after hitting the specimen
What can be used in EM to create better images? Why is this necessary?
Heavy metal stains are used to coat specimen in order to increase scatter and contrast because common cell atoms (O, H, etc) do not scatter electrons well
Why is scattering important in EM?
It creates contrast between light and dark in the images produced because they are not in colour
How do tissues need to be prepared for using an EM?
dehydrated, embedded in wax/plastic medium and sliced VERY VERY VERY THINLY
Give an example of when you would use a TEM?
If you wanted to see the cristae in a mitochondria
Give an example of when you would use an SEM?
If you wanted to see the shape of something
In TEM what can be done to provide extra contrast?
negative staining coats the grid with a substance to remove holes and drops of heavy metal stain added across the whole grid so that the specimen stands out better
In what situations would you use negative staining in TEM?
for small particulates like ribosomes, viruses, multi-subunit enzymes
How does freeze fracturing relate to EM?
You can look at freeze fractured membranes under EM and use TEM
What is the goal of SEM?
to produce an object that has the same shape and surface properties as the living state but has no fluid so it can be looked at under a vacuum
removing all water from living cells without disrupting cell structure
How is the water from living cells removed without disrupting cell structure in SEM?
water replaced with liquid transitional fluid (CO2) at specific temperature and pressure
dried specimen coated with gold –> suitable target for electron beam
e scatter to make a 3D image
