L4 - techniques - microscopy and animal models Flashcards
phase contrast microscopy
light passing through is scattered
transparent specimens
set up interference patterns in light
why cant we use light illumination
toxic
free-radical production causes growth cones to shirvel up
revolutionary technique
fluorescence
fluorescence
emits light at a different wavelength
invisible to humans
luminescent
makes its own light
fluorescent animals
budgie
stingray
scorpion
jellyfish = fluorescent and luminescent
makes its own light to create fluorescence
nobel prize GFP
2008
features of GFP
barrel-shaped
fluorophore in centre
blue light enters, green light (longer wavelength/less energy) emitted
eGFP
Enhanced (mutated) GFP
brainbow
many fluorescent proteins used
e.g. cherry RFP
useful bc greyscale is tricky to see and differentiate
advantages of phase contrast microscopy
low illumination
no labelling required
cheap
disadvantages of phase contrast
can only differentiate structures with high contrast
advantages of fluorescent widefield microscopy
allows discrimination of up to 3 or 4 fluorophores
high temporal resolution
disadvantages of fluorescent widefield
high illumination
limited depth of information
advantages of confocal microscopy
accurate 3D spatial resolution
discrimination of multiple fluorophores
automated image analysis
disadvantages of confocal
expensive
high illumination
long acquisition time
confocal microscopy
confocal laser scanning microscopy - CLSM
optical imaging technique
increases optical resolution and contrast of a micrograph
uses a spatial pinhole to block out-of-focus light in image formation.
advantages of mice as animal models
similar cortical structure
mammalian
- genes/proteins therefore conserved
disadvantages of mice
expensive
ehtical issues
advantages of frogs
vertebrate so conserved genes/proteins
produces hundreds of oocytes
disadvantages of frogs
non-mammalian
limited behavioural tests possible
advantages of using fruit fly
fast life-cycle
readily genetically manipulated
used to model Alzheimers/parkinsons
disadvantages of fruit fly
invertebrate
therefore limited conservation of proteins
advantages of using chick embryo
ethically fine - no licence needed
minimal husbandry costs - chicks are cheap
evolutionary conserved developmental events (heart beat day 2)
ready source of primary neurons
similar visual system
good for modelling congenital defects
