Total Internal Reflectance Fluorescence Microscopy- TIRFM

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Total Internal Reflectance Fluorescence
Microscopy- TIRFM
Fluorophores bound to the specimen surface and
those in the surrounding medium exist in an
equilibrium state. When these molecules are
excited and detected with a conventional
fluorescence microscope, the resulting
fluorescence from those fluorophores bound to the
surface is often overwhelmed by the background
fluorescence due to the much larger population of
non-bound molecules.
http//www.olympusmicro.com/primer/techniques/fluo
rescence/tirf/tirfintro.html
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TIRFM (or TIRF) was developed by Daniel Axelrod
at the University of Michigan, Ann Arbor in the
early 1980s. A TIRFM uses evanescent (or
vanishing) wave to selectively illuminate and
excite fluorophores in a restricted region of the
specimen immediately adjacent to the glass-water
interface. The evanescent wave is generated only
when the incident light is totally reflected at
the glass-water interface. The evanescent
electromagnetic field decays exponentially from
the interface, and thus penetrates to a depth of
only approximately 100 nm into the sample medium.
Thus the TIRFM enables a selective
visualization of surface regions such as the
basal plasma membrane (which are about 7.5 nm
thick) of cells. However, ,the region
visualized is at least a few hundred nanometers
wide, so the cytoplasmic zone immediately beneath
the plasma membrane is visualized in addition to
the plasma membrane during TIRF microscopy. The
selective visualization of the plasma membrane
renders the features and events on the plasma
membrane in living cells with high axial
resolution. TIRF can also be used to observe the
fluorescence of a single molecule, making it an
important tool of biophysics and quantitative
biology.
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TIRFM
1 Sample 4 immersion oil 2 Evanescent
wave range 5 objective lens 3 coverslip 6
emission 7 - excitation
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tirf
epifluorescence
http//physiology.uni-saarland.de/Ute_Becherer/Bec
herer_Research.html
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Fluorescent Recovery After Photobleaching (FRAP)
Fluorescence recovery after photobleaching is
used to measure the mobility of molecules. A
defined area of fluorescently labeled molecules
is bleached with intense illumination typically
using laser light Recovery of fluorescence is
recorded over time. The measured recovery of
fluorescence can be fitted with appropriate
models (diffusion, transport) leading to
diffusion constants, rate of transport or
fractions of immobilized molecules. Confocal
laser scanning microscopes are all equipped to
allow FRAP experiments.
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Initial Fluorescent signal
Bleach
Recovery
http//www.zmb.uzh.ch/resources/protocols/FRAP_en.
print.html
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FLIP (Fluorescence Loss In Photobleaching) is
used to measure the rate of protein diffusion in
the cell. The protein of interest coupled with
the GFP is introduced into cells. A small zone of
the cell is regularly bleached. Then fluorescence
outside this zone is measured. A reduction in
fluorescence outside the zone indicates a
diffusion of proteins towards the bleached zone.
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FRET (Fluorescence Resonance Energy Transfer) is
used in cellular imaging to look for molecular
interactions.  The method tests if 2 molecules
carrying each one a fluorescent group, are close
to, or far from each other. The technique is
based on the transfer by resonance towards the
acceptor, of the energy emitted after excitation
of the donor. This transfer can only take place
if the emission spectrum of the donor overlaps
the absorption spectrum of the acceptor. As this
transfer can take place only for distances lower
than 100Å, this method makes it possible to say
if 2 molecules are or not in interaction.
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Coupling of receptor
Conformational change