Microscopy & Electron Micrographs Flashcards
What illumination source does a bright field microscope have?
light
What is the maximum magnification of a BFM?
1000x - 2000x total
When are BFMs best used?
looking at LIVE specimens
getting an overview of a sample - ex. cell and tissue organization
most economical option
What is a major disadvantage of BFMs?
low resolving powers
Define resolution?
the smallest distance between two objects at which the observer can distinguish there are two objects
What is the formula for resolution?
D = 0.61 lambda/ n sin alpha
Why does resolution increase drastically with the electron microscope?
because the illumination source is not light, it is electrons and electrons have a shorter wavelength than light
shorter wavelength = better resolution
When are TEMs best used?
for looking at internal structure of cells
for viewing at very high resolution
for looking at relationships between structures
What are the disadvantages of TEMs?
samples need extensive preparation before visualizing and specimen must be dead
expensive to run
What is the max magnification of TEMs?
5,000,000 x
Describe the structure of a TEM
an electron gun (tungsten filament and anode) at the top of the column
electromagnetic condenser lenses throughout column
fluorescent screen and camera at base
specimen holder at base
Describe how a TEM works
an electron gun (tungsten filament and anode) directs electrons through electromagnetic rings which focus the electron beam through the specimen lighting up the fluorescent screen at the base
What are the two outcomes of the electron beam moving towards the specimen?
either the electrons will hit the stained specimen and reflect off, creating shadows
or the electrons will pass through the specimen and make light dots on the screen
What is the result of a TEM (what the observer sees)?
a black and white 2D image with high resolution
a slice of a specimen
In what 3 ways is a TEM similar to a light microscope?
- its condenser lenses focus beam on specimen
- specimen used is SO small (1/4 size of pinky finger nail mounted on grid)
- objective, intermediate projection lenses all magnify
How is resolution adjusted on a TEM?
by altering the voltage
higher voltage = greater resolution
What does increasing the voltage on a TEM do?
increase resolution
How is magnification adjusted on a TEM?
by adjusting the electrical current using the electromagnetic lenses
Describe the steps of preparing tissue for TEM?
- preserve fresh tissue
- dehydrate tissue using alcohol series
- coat tissue in plastic resin and let harden
- Using a microtome with a diamond knife, perform ultrathin sectioning to cut the tissue to 100nm thickness
- mount sectioned tissue onto an EM grid for staining
- stain tissue with heavy metals that coat the specimen to increase scatter of electrons = more contrast
How is an image formed from TEM?
some parts of a stained specimen may be denser than others and those dense areas will deflect electrons and create dark spots on the fluorescent screen
Parts that are not as dense will allow electrons to pass through the specimen and create light spots
The result is a 2D slice of the specimen with a black and white contrast image of the specimen
Describe how to prepare the specimen for TEM negative staining
for outlines of small particulates (ribosomes, isolated Golgi, viruses, multi-subunit enzymes, etc.)
- the grid where the specimen will be held is coated with a transparent substance to fill any holes
- drops of heavy metal stain are added to the grid and allowed to dry
- heavy metal deposits everywhere EXCEPT where the specimen is
result = more contrast between the specimen and the grid
Describe how to prepare the specimen for TEM freeze fracture
- specimen is frozen in liquid nitrogen and cracked with a cold knife to separate parts of the tissue (usually the 2 monolayers of a membrane)
- heavy metal is deposited to the frozen membrane pieces
- carbon layer deposited directly on top of the metal and acts as a cement/casting
- tissue digests/is discarded and the carbon layer can be viewed under TEM
Shows a 3D texture and improves resolution
What is the max magnification of a SEM?
~100,000x
What are SEMs best used for?
looking at the surfaces of OBJECTS (not organelles)
larger specimens
looking at objects in 3D
What are a few disadvantages of SEMs?
resolution is not as high as TEM
specimen must be dead and coated in metal before visualizing
expensive to run
How do tissues need to be prepared for viewing in an SEM?
tissue must be dehydrated (by sublimation under pressure) but its shape and structures must be maintained
dried specimen must be coated in gold to make it suitable for electron beam
Describe how an SEM works
an electron beam SCANS the surface of the gold-coated specimen
as the metal evaporates, the atoms condense on the surface of the specimen
electrons are reflected off surface and detected to create a 3D image
Which of the 3 microscopes can visualize live specimen?
light microscope
What kind of lenses do the 3 microscopes use?
light: glass
TEM & SEM: magnets
What kind of images do each of the 3 microscopes produce?
light: coloured images
TEM: 2D black and white slices
SEM: 3D black and white images
What kind of stains can be used in each of the microscopes?
light: optional coloured stains
TEM: required heavy metal stains to stick to cellular structures
SEM: required gold coating over surface of specimen
What is a major advantage of light microscopy?
live specimens can be visualized and colour and movement can be observed
What is a major advantage of TEM?
the best resolving power
What is the major advantage of SEM?
it produces a 3D image of a specimen
What is the benefit of using all 3 microscopes?
Looking at the 3 different micrographs will each give us different information about a specimen and combining all 3 will give us a better understanding of the specimen
