A2.2.3

• Immunofluorescence

  • Antibodies that bind to particular antigens in the cell are produced
  • Fluorescent markers of different colours are linked to these antibodies
  • A multicoloured fluorescent image can be produced which shows where the antigens are located
• Fluorescent stains

  Coloured substances that bind to some chemicals but not others
• Some fluorescent stains absorb ultraviolet light and re-emit it as blue light
• Fluorescence microscopes

  Light emits a single wavelength which is absorbed and re-emitted by the sample generating particularly bright images
• Fluorescence

  Substance absorbs light and then re-emits it at a longer wavelength
• Some of the ice is removed by vaporisation to enhance the texture of the surface
• The technique is used to produce images of surfaces within cells
• The replica is removed from the frozen sample and can be examined using an electron microscope
• The freeze fracture process gives a unique image of this part of cells
• Freeze-fracture electron microscopy

  A technique used to produce images of surfaces within cells
• A steel blade is used to fracture the frozen sample
• Steps in the freeze-fracture electron microscopy technique

  • Samples are put into liquid propane at -190°C
  • A steel blade is used to fracture the frozen sample
  • Some of the ice is removed by vaporisation to enhance the texture of the surface
  • A vapour of platinum or carbon is fired onto the fracture surface to form a coating
  • The replica is removed from the frozen sample and can be examined using an electron microscope
• Uses of the freeze-fracture electron microscopy technique

  • Images of surfaces within cells
  • Unique image of the weakest point of cells
• A vapour of platinum or carbon is fired onto the fracture surface to form a coating
• The weakest point is usually the middle of membranes (between two phospholipids)
• When these images were first produced they led to a fundamental change in theories about membrane structure
• Cryogenic electron microscopy involves freezing samples prior to viewing, which allows for the determination of molecular structures at near atomic resolution without requiring the crystallisation of the specimen.
• Freeze fracturing was used to demonstrate the presence of integral membrane proteins within the plasma membrane.