Exam 2 Confocal Microscopy - Lecutre 8: FRAP/FLIp, caged compounds, optical highlighters, laser capture microscopy

types of diffusion measurements

FRAP

FLIP

caged compounds

optical highlighters

FRAP-fluorscence revoery after photobleaching

measure rate of fluorscence recovery in photobleached region

circle small region of interest and then photobleach and monitor over time

fluorochromes can amigrate to bleacher spot and it will recover

mobile fraction

percentage of protein the can move around in sample

close to 100% - unrestricted movement

close to 0 - movement restricted

diffusion measurements

the rate of protein movement within a cell

D increases

-decrease in viscosity

-involved in active transport

D decreases

-increase in viscosity

-formed an aggregate

-interacting with large or fixed molecules

FLIP - Fluorescence loss in photobleaching

measure rate of fluorescence loss at a location away from photobleaced region during continuous bleaching

remote area should see decrease in intensity since bleaching is constant and there will be no movement

what does FLIP determine

if intracellular compartments or membranes are connected

loss of fluorescence in area A indicates that A and B are connected and the protein may freely travel from A to B

no change in intensity in area A indicates that the path from A to B are not connectedcage

caged compounds

photoactivatable probes

biologically inactive until exposed to a pulse of UV light

UV light changes the chemical structure of the probe into an active form

advantages with caged compounds

reagent is not active when added to the cells

with the laser, the exact location can be defined

with the laser, the exact time for release can be defined

multiple locations and time points can be viewed using one tissue or culture dish

optical highlighters PAFPS

fluorescent proteins that change color or emission intensity over time or in response to photoactivation

laser capture microscopy (microdissection)

allows laser to cut specific ares of tissue / cellular components

steps of laser capture microscopy

1) trace region of interest

2) isolate the region by cutting the sample along the drawn line using a focused 355nm laser

3) catapult the isolated sample into the cap by a pulse of the defocused laser beam

ways to section prep

parafin wax embedded sections cut by microtome (requires high temperature)

frozen sections cut by cryostat

epidermal peels

live cells in culture

PEN membrane slides

sections are attached to a thin transparent membrane

chemically inert

enhances cut and supports tissue section

what to do with microdissections

DNA analysis, RNA analysis, protein analysis