Lecture 1 Flashcards
Preparation For Light Microscopy
- Fixation- Formalin (formaldehyde) which reacts with tissues to cross link proteins and other substances
- Dehydration- Alcohol or acetone
- Embedding- paraffin or plastic
- Sectioning- generally 2-10 um thick
- Mounting- glass slide
Hematoxylin and eosin (H and E)
H has positive charge and stains negatively charged structures (nucleus). E is negatively charged and stains positively charged substances (cytoplasm).
Periodic Acid Schiff Stain
For carbohydrates like sugars (glycogen and glycoproteins)
Aldehyde Fuchsin
Stains Elastic Fibers and b cells of the pancreatic islets. Connective tissue
Orcein
Stains collagen fibers
Silver Stain
Stains golgi apparatus and reticular fibers
Resolution of Light Microscopy
- about 0.2 microns.
- useful Magnification of about 1,500 times
Resolution of Transmission Electron Microscope
- about 1-1.45 nanometer
- useful magnification greater than 500,000 times
Resolution of Scanning Electron Microscope
- about 2 nm
- useful magnification is greater than x100,000
Preparation for TEM
- Fixation - with glutaraldehyde that fixes proteins and nucleic acids followed by osmium tetroxide that fixes lipids.
- Dehydration
- Embeddign: plastic such as Epon
- Sectioning: 20-100 nm thick using ultramicrotome using glass or diamond knife
- Mounting: wire mesh
- Staining: heavy metals are used to enhance electron density
Freeze Fracture
- method of specimen preparation
- follows the hydrophobic region of the membrane exposing 2 fracture faces
- the E face- inwardly facing outer half
- P face- the outwardly facing inner half
Preparation of Scanning Electron Microscopy
- Fixation and dehydration - same as TEM
- Dried using the critical point method to avoid drying artifacts
- mounting - glued to a metal stub
- coated with metal (gold/palladium) so it will enhance emission of electrons
