Principles of microscopy Flashcards
Antonie van Leeuwenhoek
built microscopes with x300 lenses
Components of a light microscope:
a) condenser lens -focuses light onto specimen
b) objective lens to collect light scattered by specimen
c) eye piece
Limit of resolution
Human eye = 200um (0.2mm)
Light microscope - >0.2um beetween two structures
Tissue preparation
-needs to be firm or -chemically fixed or -cell suspensions (artificial matrix)
phase contrast microscopy
differences between refractile index of cells and water environment causes shift in phase of wavelengths and creates image – e.g for buccal smear or cell cultures
Fixation:
- human tissue -immertion fixation
- use of formaldehyde or gluteralgehyde
- often use of second fixative-osmic acid, to preserve lipids
Dehydration:
- removal of water content of cell
- use of alcohol for paraffin embedding
Embedding:
- prevents specimen collapsing-medium has fluid phase but has potential to harden allows cutting the glass or metal knives on microtome
- tissue blocks in resin/paraffin
Sectioning
- paraffin wax or frozen sections approx 7um thick
- diamond knives can get 1um sections
- suitable for transmission electron microscopy
H&E Stain
most common stain
Haematoxylin
basic dye with +ve charge
- binds to -ve species (eg phosphate groups in nucleic acids)
- purple/black cell nuclei
Eosin
-acid dye binds to +ve charged groups
(amino groups in proteins)
eg. collagen fibres, general cytoplasm=pink
van Geison method
- stains collagen pink
- muscle=yellow
- commonly used in combination with a stain for elastic fibres
Periodic acid-Schiff (PAS)
- carbohydrates
- eg mucous cells of the stomach are strongly PAS +ve
Alcian blue
-identifies mucous secretion cells
