Flashcards in Investigative Techniques Deck (22):
What are the requirements to image tissues by light microscopy?
• Need to preserve the tissue to prevent it from rotting
• Need to embed the tissue in a substance that allows it to be sliced
very thinly - down to 5 micrometers thinness (using microtome)
– Melted Paraffin that sets hard when cooled
• Need to stain the tissue so that we can see cell components
– Most commonly Haematoxylin and Eosin
What do we use to stain tissue for light microscopy?
1. Haematoxylin only
- Haematoxylin stains the nucleus blue most strongly
2. Eosin only
- Eosin stains the cytoplasm and extracellular matrix pink most strongly
3. H and E
- The nuclei are clearer and there is more detail in the cytoplasm
- Overall purple colour
How would you carry out a frozen section?
- The surgical specimen is placed on a metal disc which is then frozen rapidly to –20 to –30 °C.
- Tissue become rock-hard.
- Cryostat (microtome inside a freezer) cuts the frozen specimen
- Stained with hematoxylin and eosin.
- Sample preparation is more rapid than traditional technique (10 minutes vs 16 hours)
- Technical quality of the sections is much lower
Name a suitable method to test for gout.
- Polarised light microscopy of joint fluid
- Uric acid precipitates in joint to form urate crystals
- Yellow and needle shaped under polarising microscope
- Blood test results can be misleading. Possible to have signs and symptoms of gout, but don't have unusual levels of uric acid in their blood (as it is within their joints).
What is Immunohistochemistry?
- Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
- Then attach a secondary antibody that will change colour upon staining or a fluorescent tag.
What is autoradiography?
- In autoradiography a photographic emulsion is used to visualise molecules labelled with a radioactive marker
- An autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance.
What is the magnification of a modern light microscope?
- Magnification = 1000x
What is the distance between resolvable points of a modern light microscope?
Distance between resolvable points is 0.2 micrometers.
What is 'resolution'?
The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities.
What contributes towards good resolution?
- High frequency
- Short wavelength
- The image of two particles cannot be seen individually if it is smaller than the wave length
What is the wavelength of a modern light microscope?
400 nanometers in wavelength.
Why can a transmission electron microscope not be used for living organisms?
- Uses an electron beam generated in a vacuum
- Living organisms cannot survive inside a vacuum
How does the resolution of an electron microscope compare to that of a light microscope?
- Wavelength is 1 nanometre compared to light microscopy which uses 400 nanometres.
- The resolution of an EM is therefore 400x that of light microscopy
How does the magnification of an electron microscope compare to that of a modern light microscope?
- Magnification is typically 250,000 x. Light microscopy has
magnification of 1,000 x.
- Therefore EM has a magnification 250 x that of light
How does a TEM work?
- TEM uses electromagnetic lenses to focus the electrons into a very thin beam
- The electron beam passes through the tissue.
- Those portions that the beam have passed through appear bright, those portions that have absorbed or scattered electrons appear dark
What is freeze fracture EM?
1. Tissue is frozen to -160 degrees C
2. Tissue is fractured by hitting with a knife edge.
The fracture line passes through the plasma membrane exposing its interior which can then be imaged.
What is scanning electron microscopy?
• The electrons are reflected back from the surface and received by a cathode ray tube (like a TV)
How does fluorescent microscopy work?
- Specimen has flurofors (fluorescent molecules) attached to it
- Flurofors emit light when radiated by specific light wavelength
- Different colours show different cell components or different types of cell within a tissue
- Useful for biomedical research e.g. Differentiation of different cancer cell types
Describe how confocal microscopy works
- Same technique as CAT scanning (computerised axial tomography)
- Beam of light illuminates specimen, mirrors and then reflects light towards pinhole opening in front of the camera
- Only light from single thin slice passes through the pinhole
- Blocking the rest of the light creates a sharper image
- Combined with fluorescent imaging and dyes to locate specific structural regions
- Allows for construction of 3D imagine
- Images captured in multiple planes
- Combined image data
- Images taken over period of time at regular intervals to create time lapse (of living organisms)
Describe how confocal microscopy is advantageous over a normal microscope
- Normal microscopes require tedious cutting processes and prevent imaging of living organisms
Whats a monoclonal antibody?
- All originate from a single, hybrid cell line, hybridoma
- Hybridoma = combination of B cell and myeloma (cancerous plasma cells)
- Cant produce new antibody chains - highly specific
- Polyclonal antibodies produce a variety so the antibodies can bind do different epitopes
- Because they are specific – less side effects