Flashcards in Investigative Techniques Deck (17):
Which technique is often used to view tissues?
What is the magnification of modern light microscopy?
- Magnification = 1000 x
- Distance between resolvable points = 0.2 um
What are the requirements to image tissues by light microscopy?
1. Preserve tissue to prevent rotting - formalin
2. Embed tissue in a substance that allows it to be sliced very thinly (down to 5 um thinness) - melted paraffin that sets hard when cooled.
3. Stain tissue to see all cell components - most commonly haematoxylin and eosin.
Which machine is used to cut tissue slices very thinly?
What do haematoxylin and eosin each stain most strongly?
- Haematoxylin: stains the nucleus blue most strongly
- Eosin: stains the cytoplasm and extracellular matrix pink most strongly
Which sample preparation method could be used that is faster than the traditional histology technique?
Cryosection using a cryostat (microtome inside freezer):
- specimen frozen to -20 to -30 degrees
- much more rapid: 10 min vs 16 hrs
- much lower technical quality of sections
What are the different types of light microscopy?
Polarised light microscopy:
- contrast-enhancing, can evaluate composition and 3D structure of specimens.
- enables reconstruction of 3D structures from sets of images obtained at different depths
What is the difference between immunofluorescence and indirect immunohistochemistry?
- Primary mAb labelled with fluorescent tag binds target protein and emits signal.
- Primary mAb binds target protein.
- Secondary mAb labelled with enzyme produces coloured product on binding primary mAb.
What is autoradiography?
- Radioactive marker injected into live animal/cell culture.
- Histological section coated with photographic emulsion - allows visulalisation of marker.
Give an example of a radioactive marker.
Why can light microscopy not be used to visualise organelles?
- Wavelength of beams is not small enough.
What kind of microscopy is required to view organelles?
Transmission electron microscopy (TEM)
What is the principle of TEM?
- Uses an electron beam generated in a vacuum.
- The electron beam passes through the tissue:
~ portions that the beam have passed through appear bright
~ portions that have absorbed of scattered electrons appear dark
Compare the wavelengths and magnifications of LM and TEM.
- TEM has 1 nm wavelength; LM has 400 nm wavelength. So TEM has 400x resolution of LM.
- TEM has magnification of 250,000x; LM has magnification of 1,000x. So TEM has 250x magnification of LM.
What are the requirements for TEM?
1. Fix with glutaraldehyde.
2. Embed in epoxy resin.
3. Stain (e.g. Osmium tetroxide)
4. Use microtome with diamond knives.
Name other types of electron microscopy.
1. Freeze fracture EM
- Tissue is frozen to -160 degrees and fractured by hitting with a knife edge.
- Fracture line passes through the plasma membrane - exposes interior which can be imaged.
2. Scanning EM
- Electrons are reflected back from the surface and received by a cathode ray tube.