L2 microscopy

• structure is always related to

  function
• why use microscopy?

  • To visualize small objects
  • visualize bacterial growth
  • in cell culture (count cells)
  • location of proteins
  • host-pathogen interactions
  • abundance of proteins after stimulation
  • proliferation or death markers
• define magnification

  the ratio of an object's image size to its real size
• define resolution

  the measure of the minimum distance of 2 distinguishable points
• define contrast

  visible differences in brightness or colour between parts of the sample
• bright field microscope works by:

  • light passes through the specimen so specimen must be at least partially transparent
  • light comes from light source below the stage and the condenser lenses
• advantages of using light microscope

  image living cells
• disadvantage of using light microscope

  limited resolution (best being 0.2 microns)
• sample types:

  • whole mounts
  • tissue sections
• preparing tissue sections: (light microscopes)

  1. fixation, preserves structure (chemical fixatives to prevent autolysis)
  2. dehydration and clearing
  3. embedding (molten wax added to specimen)
  4. sectioning (cutting into 5 microns thick using microtome and collected onto slides)
  5. staining (wax is removed and dye used)
• types of dyes:

  • Eosin (cytoplasm)
  • haematoxylin (nuclei)
• how does advanced light microscopy work?

  light phase shifts using polarised light
• how does fluorescent microscopy work?

  using fluorescent dyes or proteins that emit light when excited by specific wavelengths of light, allowing for the visualization of specific molecules or structures within a sample.

  target protein bound by primary antibody which is bound by fluorescent secondary antibody which the fluorescent signal detects
• how does confocal microscopy work?

  using laser scanning through pin hole
• advantage of confocal microscopy

  • generate 3D images of living cells
  • remove out of focus images
  • can look inside thick specimens
• what is super resolution?, how does it work?

  gathers light from individual fluorescent molecules and records their position

  combining information from these individual molecules breaks the resolution limit
• difference between canning and transmission electron microscopy?

  transmission-inside cell

  scanning- surface of cell
• why do electron microscopes need a vacuum?

  electrons have poor penetrating power, and focused using magnetic fields
• electron microscope resolution

  0.08 nm
• preparing tissue sections (transmission electron microscopy):

  1. fixation (glutaraldehyde-protein crosslinking, then osmium tetroxide- lipid crosslinking)
  2. dehydration (in ethanol)
  3. embedding (plastic resins e.g. epoxy)
  4. sectioning (50nm thick, cut using ultramicrotome)
  5. staining (heavy metal stains e.g. lead to improve contrast)
• difference with scanning electron microscope tissue sectioning:

  critical point drying-> technique which allows ethanol to be removed from sample, minimises shrinkage

  coating specimens-> thin layer of gold to protect from electron beam damage
• what is cell fractionation?

  allows major organelles to be individually separated out