Total Internal Reflectance Fluorescence MicroscopyTIRFM
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/techni
ques/fluorescence/tirf/tirfintro.html
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 glasswater 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.
TIRFM
1 – Sample
2 – Evanescent wave range
3 – coverslip
4 – immersion oil
5 – objective lens
6 – emission
7 - excitation
epifluorescence
http://physiology.unisaarland.de/Ute_Becherer/Becher
er_Research.html
tirf
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.
Initial Fluorescent signal
Bleach
Recovery
http://www.zmb.uzh.ch/resources/protocols/F
RAP_en.print.html
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.
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.
Coupling of receptor
Conformational
change