class 29 - techniques fluorsecent microscopy

fluorescent microscopy

filters block other wavelenghts of light, to limit excitation and emission to only fluorescent molecules of interest

how to run in vivo

step 1: add fluorescent
step 2: excite and measure emission

step 1: add fluorescent

stain/dye celll or tag proteins

GFP

from jellyfish, intrinsically fluorescent

DAPI

can color cellular components, binds to AT rich regions of DNA, staining the nucleus blue

localization question

where are my proteins?

localization

proteins are made fluorescent via dyes and protein tags, identify boundaries, if in the same area use different colors

FISH abbreviation

fluorescent in situ hybridization

in situ

in place

FISH

fluorescent probe is attached to a DNA/RNA fragment, probe is mixed into cells, complementary base pairing hybridizes probe with DNA, signal = where DNA is in cell

Alexa fluoro

can stain anything in any color because it binds to antibodies, and antibodies can bind to anything

FRET abbreviation

fluorescent resonance energy transfer

FRET

technique uses 2 fluorophores with overlapping emission (donor) and exitation (acceptor) ranges

FRET - if proteins are not interacting

accpetor is not excited, light emitted within donor’s emission spectrum

FRET - if proteins are interacting

donor emission will be absorbed by acceptor, light emitted will be within acceptor’s emission spectrum

FRAP abbreviation

fluorescent recovery after photobleaching

FRAP

membrane proteins are fluroescently labelled, a single area is bleached with high intense light, membrane components diffuse normally until bleached patch is covered again, rate of recovery = fluidity