Microscopy Flashcards
What are the types of unstained light microscopy techniques?
Bright Field, Phase Contrast and Differential Interference contrast
What is Bright Field?
Normal, no special techniques
What is Phase Contrast?
Uses same size phase condenser to enhance the differences between wavelengths. This is a very simple technique
What is Differential Interference Contrast?
Very complicated
What is Kohler Illumination?
The technique for optimising resolution and creating even sample illumination
What is sample fixation?
Preserving the structure and contents of tissue.
What is an example of a fixation agent?
Formaline
What is the dehydration step?
Dehydration is done to remove all liquid and ‘dry’ the sample. This often uses ethanol.
What is used to remove the dehydrating agents?
A clearing agent such as chloroform
What is embedding?
Solidifying a sample so that it can be cut
What are the two main types of embedding?
OCT freezing and molten parrafin wax.
What are the benefits of using OCT freezing?
Allows the antigenic properties to be preserved- used in immunohistochemistry.
What are the benefits of wax embedding?
Allows the structure of tissue to be retained- used in pathology
What is a cryostat?
Freezing and microtome machine (5-10um)
How thin does a microtome cut to?
4-30um
What does haematoxylin stain?
Nuclei dark blue and all other structures pink/purple
What does Safranin O and Fast Green stain?
Cartilage red and muscle green/blue
When would you use Safranin O/Fast Green stain?
In the study of osteoarthritis
What does Masson trichrome stain?
Collagen blue, nuclei black and muscle/cytoplasm/keratin red
What does Vanhoff Gierson stain?
Elastic fibres and nuclei blue/black, collagen red and other structures yellow
When would you use Masson trichrome stain?
Liver, Kidney or tumor biopsy
When would you use Vanhoff Gierson stain?
Elastic structures such as blood vessels and in tumour invasion determination
What is a flurophore?
A substance that emits light at a different wavelength to that at which it absorbed it
What is stoke shift?
Difference between the absorbed and emitted wave lengths
What is a filter cube?
A cube which allow specific wave length to hit the target and a detector to absorb the emission from the target without interference from the original source.
What is direct staining?
An antibody bound to the stain directly binds to the target.
What is indirect staining?
The antibody bound to the stain binds to another antibody, which itself has bound to the target
What does Wheat Germ Agglutinin stain?
Plasma membrane
What does Phalloidin stain?
Actin
What does DAPI stain?
Double-stranded DNA
What is the extinction coefficient?
The intrinsic property of a substance to absorb/reflect certain waves lengths
Does the Formaldehyde preserve structure or antigenicity?
Structure
Does the Methanol fixing agent preserve structure or antigenicity?
Antigenicity
What is permabilisation?
‘poking’ holes in the cell to allow the stain to enter. An example of this is Tritin X.
What is blocking?
Blocking undesirable antibody binding site
What is often used as a blocking agent
Bovine Serum Albumin
What is confocal microscopy?
Using a thin ‘slice’ of the visual field reduces light interference and increases resolution. It also allows for 3D images to be constructed
What is widefield?
Normal microscopy
When do you use transmission electron microscopy?
For small samples and to their ultrastructure
When do you use a scanning electron microscope?
For larger samples and to see their surface in detail
What is a conventional SEM electron source?
A plate is heated up until it produces electrons. This is more common and simple, but as the electrons are not as focused it cannot give the ‘zoom’ of the other type (50,000)
What is a field emission SEM electron source?
A strong electromagnetic field is created with a current to push electrons into the beam. The electron beam is much more powerful and therefore can increase the zoom and resolution (1,000,000x)
What is backscatter vs non-backscatter?
Non-backscatter uses the secondary electrons emitted from the sample to create an image whereas backscatter uses the electrons which are reflected from the sample to create the image
What must happen when samples are viewed under SEM High Vacuum?
They must be gold-palladium plated
What does Variable pressure SEM allow for?
Uncoated fresh samples to be viewed
What is the gold-palladium coating known as?
The sputter coat
Is the sputter coat applied first or second?
It is the secondary fixing agent
When fixating TEM, which methods can be used?
Chemical fixation or critical point freezing fixation
What is an ultramicrotome?
A microtome that sections to 90nm
With TEM, what can be used as the primary fixator?
2.5% glutaraldehyde
In TEM, what can be used as the secondary fixator?
1% Osmium tetroxide
What is used for critical point drying?
Hexamethyldisilizane
