Microscopy Flashcards
(9 cards)
What does the objective lens do
collects a cone of light rays to create an image
Condenser lens?
Focuses a cone of light rays onto each point of the specimen
Stains can bind with some specificity - depending on physiochemical properties
Acidity - DNA, lysosomes, pectins
Alkalinity - mitcochondra matrix, chloroplast stroma, hydrophobicity - lignin, lipid droplets, membranes
Specific chemical interactions - protein, starch, polysaccharide
Give an example of where enzyme activities can be used to stain specific structures
Peroxisomes can be stained with 3,3’ diamino benzidine (DAB) due to high peroxidase content
What are the advantages of fluorescent labels over chromogenic labels
more sensitive less background higher contrast Better for labelling multiple structures more easily quantified
What are the 3 main types of flurorescent label
- Small organic chemicals that associate with particular structures e.g. DAPI
- Antibodies coupled to a fluorescent molecule eg Cy5
- Genetically encoded fluorescent protein tag
Small organic chemiacals
e.g. 4’,6-diamidino-2-phenylindole (DAPI) synthetic compound binds the minor groove
Rhodamine-phalloidin binds F-actin, natural toxin coupled to a fluorescent rhodamine
Phalloidin toxin from death cap fungus
Antibodies - why are they used?
Most biological molecules are not specific enough for the design of specific chemical probes - e.g. for individual proteins
Why are antibodies so brilliant
- Bind to foreign molecules that enter intercellular fluid
- Enormous diversity in the variable regions which allows high specificity
- Purified protein trigger production of complementary antibodies
- Each antibody recognises a different small region of molecule (Epitope) - 6-10a.acids
Can be purified form the serum of an immunised animal and used as specific probes for individual molecules
