Electron microscopy Flashcards
How does the resolution in electron microscopy compare to light microscopy?
Light: 200nm, electron: 0.1nm.
What is the basis of the electron microscope?
Electrons are emitted from a filament and accelerated in an electric field. A condenser lens focuses the electron beam.
How do TEM and SEM differ?
In TEM, electrons scatter or hit a fluorescent screen at the bottom of the microscope, whereas in SEM electrons are focused on a metal coated speciment and the electrons are then collected by a detector. The electrons are already scattered before they hit the specimen in SEM.
Why can specimens not be alive in electron microscopy?
Due to the high pressure vacuum that needs to be present.
What method is used to view samples in TEM?
A transparent formvar film is floated off a glass slide onto the surface of water in a petri dish. Grids at the bottom of the fish are lifted out of the water and the film will be deposited on them.
Why is a support film needed in TEM?
To enhance stability and conductivity when exposed to the electrons.
How can sample contrast be increased in TEM?
Negative staining using heavy metals such as lead, uranium, tungsten and gold. Shadowing can be used where heavy metal is evaporated from a wire in a vacuum chamber to cast a shadow on the adjacent sample.
How does shadowing work?
The sample is spread on a mica surface and then dried. The sample is coated with a film of heavy metal and the remaining biological material is then dissolved with acid and bleach.
How can protein amino groups be preserved during sample preparation?
Using glutaraldehyde.
How can proteins and lipids be preserved during sample preparation?
Using osmium tetroxide.
How can membranes be preserved during sample preparation?
Using potassium permanganate.
Why do samples need to be preserved in sample preparation?
As samples are usually around 70% water and they would boil in the EM vacuum.
How can the samples be dehydrated?
Using increasing concentrations of ethanol in different steps.
What is the purpose of embedding in TEM?
To produce blocks that are suitable for ultra-thin sectioning and still preserve the fine structure of the specimen.
How can samples be embedded?
The sample should be placed in a BEEM capsule and infiltrated with un-polymerised epoxy resin, Epon or Araldite. The resin is them polymerised in the capsule and the resin block should be removed.
How can tissues be sectioned?
Using an ultra-microtome to produce ultra-thin sections (50-90nm thick). These are then picked up on formvar coated grids.
How do fixatives differ?
They can vary in their rate of penetration, their ability to fix different molecules and can cause shrinkage or swelling of tissues.
How can cell components be localized in light microscopy?
Using histochemical dyes, antibodies linked to fluorescein isothiocyanate, GFP and chromogenic enzyme substrate.
How can cell components be localized in electron microscopy?
An antibody linked to colloidal gold, enzyme localization by linking product to heavy metal e.g lipases with Pb and enzyme localization if products are electron dense e.g. peroxidase.
How can proteins be detected in electron microscopy?
Antibodies and Protein A-gold can be used. Protein A-gold will bind to the antibodies added and can locate teh molecule of interest.
How can different sizes of colloidal gold be used to identify different molecules?
The different sizes can be used to identify different molecules simultaneously.
What can be used to identify phenols?
Peroxidase - it converts phenols to electron dense products.
How does SEM work?
Electron beams scan the specimen and cause it to emit electrons to give a detailed image.
What is the magnification range of SEM?
x10 to x100000
What is the resolution of SEM?
<10nm
What are some other key features of SEM?
There is a great depth of focus, the specimen can be rotated and tilted and large, irregular specimens can be viewed.
What kind of images are produced with SEM?
3D images that can be artificially coloured.
What is the preparation for samples in SEM?
Similar to TEM, but delicate specimens need to be dehydrated.
What is the process for critical point drying?
The species needs to be dehydrated in an ethanol-water series, replaced with amyl acetate and subjected to liquid CO2 under pressure and then raise the temperature as liquid CO2 will convert to a gas to leave a dry specimen.
What are the problems with critical point drying in SEM?
There may be shrinkage or the removal of substances in organic solvents such as leaf wax.
How can the problems with critical point drying in SEM be overcome?
Cryo-SEM.
What does Cryo-SEM mean?
Low temperature scanning electron microscopy.
Why is cryo-SEM favoured over the normal SEM?
The sample retains water as it is flash freezed in liquid nitrogen. There is no exposure to fixatives or solvents, and the preparation time is short.
What are the steps in cryo-SEM?
Mount the specimen on a holder, flash freeze in liquid nitrogen, transfer to a vacuum chamber of SEM (-100C), warm the stage above -96C to sublimeaway any surface ice crystals and then coat with metal and observe the specimen.
