Investigative Techniques Flashcards Preview

ESA 1 - Body Logistics > Investigative Techniques > Flashcards

Flashcards in Investigative Techniques Deck (17)
Loading flashcards...
1
Q

Which technique is often used to view tissues?

A

Light microscopy

2
Q

What is the magnification of modern light microscopy?

A
  • Magnification = 1000 x

- Distance between resolvable points = 0.2 um

3
Q

What are the requirements to image tissues by light microscopy?

A

Need to:

  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.
4
Q

Which machine is used to cut tissue slices very thinly?

A

Microtome

5
Q

What do haematoxylin and eosin each stain most strongly?

A
  • Haematoxylin: stains the nucleus blue most strongly

- Eosin: stains the cytoplasm and extracellular matrix pink most strongly

6
Q

Which sample preparation method could be used that is faster than the traditional histology technique?

A

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
7
Q

What are the different types of light microscopy?

A

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

8
Q

What is the difference between immunofluorescence and indirect immunohistochemistry?

A

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.
9
Q

What is autoradiography?

A
  • Radioactive marker injected into live animal/cell culture.

- Histological section coated with photographic emulsion - allows visulalisation of marker.

10
Q

Give an example of a radioactive marker.

A

Iodine

11
Q

Why can light microscopy not be used to visualise organelles?

A
  • Wavelength of beams is not small enough.
12
Q

What kind of microscopy is required to view organelles?

A

Transmission electron microscopy (TEM)

13
Q

What is the principle of TEM?

A
  • 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
14
Q

Compare the wavelengths and magnifications of LM and TEM.

A
  • 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.
15
Q

What are the requirements for TEM?

A
  1. Fix with glutaraldehyde.
  2. Embed in epoxy resin.
  3. Stain (e.g. Osmium tetroxide)
  4. Use microtome with diamond knives.
16
Q

Name other types of electron microscopy.

A
  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.
17
Q

Which imaging technique relies on nuclear magnetic resonance?

A
  • 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.