ways of imaging nervous system

• light microscopy

  use visual light to visualise cells and tissue

  can see astrocytes and cortical neurones

  
• Fluorescent Microscopy (wide field)

  tissue with fluorescent tag

  shine particular wavelength of light - excitation wavelength- absorbed by tag

  excites the electrons = higher energy state

  emission of wavelength of light

  
• confocal microscopy

  excitation light from laser shone onto sample

  excitation pinhole - narrow beam of light onto sample

  light absorbed by fluorescent tag in particular plane of field

  electrons are excited

  emission light released

  filter selective for the light emitted from specific plane

  
• electron microscopy

  SEM - 3D image

  electrons bounce off surface

  TEM - internal structure

  electrons pass through sample
• Tissue preparation for staining

  tissue harvested and preserved - snap frozen or chemically fixed

  tissue is cut

  frozen or embedded in wax or resin

  tissue is mounted onto glass slide - snap frozen tissue is chemically fixed

  staining

  coverslip

  image
• H&E stain

  Hematoxylin (H)

  • dark blue/violet
  • positively charged
  • binds to negatively charged DNA and RNA in nucleus
  • binds to RNA via RER

  Eosin (E)

  • pink
  • negatively charged
  • binds to positive amino acid side chains in cytoplasm

  
• Nissl stain

  Nissl

  • dark blue/violet
  • positively charged
  • bind to negative RNA/DNA in nucleus
  • binds to negative RNA in RER

  
• Golgi stain

  black

  impregnates tissue with potassium dichromate and silver nitrate

  
• osmic acid

  Black

  binds to lipids- myelin (fatty sheath)

  
• Immunohistochemistry

  antibodies -

  • fluorescent
  • enzyme complex - produces a dye precipitate in presence of its substrate
  • binds to specific protein (antigen)

  making the antibody:

  • antibody raised against antigen of interest
  • antigen introduced to animal
  • stimulates immune response
  • collect and purify antibody

  
• direct immunochemistry

  single antibody

  binds directly to antigen

  tagged with fluorophore or enzyme complex

  
• indirect immunochemistry

  TWO antibodies - primary and secondary

  primary:

  • binds directly to antigen

  secondary:

  • binds to the primary antibody
  • tagged with fluorophore or enzyme complex

  
• antibody nomenclature

  structure: