Which technique is often used to view tissues?
Light microscopy
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?
Need to:
- Preserve tissue to prevent rotting - formalin
- 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.
- Stain tissue to see all cell components - most commonly haematoxylin and eosin.
Which machine is used to cut tissue slices very thinly?
Microtome
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.
Fluorescent microscopy
Confocal microscopy
- enables reconstruction of 3D structures from sets of images obtained at different depths
Immunofluoresence microscopy
What is the difference between immunofluorescence and indirect immunohistochemistry?
Immunofluoresence
- Primary mAb labelled with fluorescent tag binds target protein and emits signal.
Indirect immunohistochemistry
- 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.
Iodine
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?
- Fix with glutaraldehyde.
- Embed in epoxy resin.
- Stain (e.g. Osmium tetroxide)
- Use microtome with diamond knives.
Name other types of electron microscopy.
- 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. - Scanning EM
- Electrons are reflected back from the surface and received by a cathode ray tube.
Which imaging technique relies on nuclear magnetic resonance?
- Magnetic Resonance Imaging (MRI)
- Based on nuclear magnetic resonance (NMR) spectroscopy.
- Provides detailed info about the structure, dynamics, reaction state and chemical environment of molecules.