The best people to answer specific questions are out there in labs. There is a confocal bulletin board that you can subscribe to where questions and answers are posted. This bulletin also has a very useful searchable archive covering a variety of subjects relating to microscopes and attachments, as well as sample preparation and choice of dye. Follow this link to get to this site.
 http://listserv.acsu.buffalo.edu/archives/confocal.html
FRAP - Fluorescence Recovery After Photo-bleaching
In a FRAP experiment, a region with fluorescent molecules is irradiated or photo-bleached with laser light. This results in the fluorescent molecules inside that region to become non-fluorescent. The recovery part of this experiment is the subsequent redistribution of fluorescent and bleached molecules throughout the volume. This gives information on their mobility.
Using FRAP, one can determine the mobility of fluorescently tagged proteins in living cells. FRAP allows quantitation of a number of 'dynamic parameters' such as diffusion coefficient, immobile fraction and binding or residence time.
- FRAP can be applied to one photon or multi-photon
- Slow FRAP takes seconds and fast FRAP takes micro- to milli-seconds
- The amount of light that recovers (returns) relative to the amount of light that was there before the photobleaching is the percent recovery.
- The speed with which the fluorescent molecules migrate back into the photobleached area is a measurement of the "diffusional mobility" which is usually called lateral mobility.
| References: |
| J Cell Sci 112(19) 3299-3308 (1999) |
| Current Biol 2000 10: 1-7 |
| FASEB J Dec 14, 2001 |
| J Cell Sci, 112 (20) 3463-3475 (1999) |
| J Cell Sci 113, 81-89 (2000) |
| The Journal of Cell Biology, Volume 153, Number 7, June 25, 2001 1341-1354 |
| Nat Cell Biology Vol 4, April 2002, E91 |
| http://www.the-scientist.com/ |
| http://www.eur.nl/ |
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FRET - Fluorescence Resonance Energy Transfer
This technique can deliver information about distance between molecules. Molecules are labelled with two fluorophores where the emission spectrum of the donor overlaps the absorption spectrum of the acceptor.
Non-radiative energy passes from the excited DONOR molecule to the ACCEPTOR molecule which then emits fluorescence. The fluorophores must be extremely close to one another for this to happen ( < 0.1mm).
This should not be confused with photonic energy being absorbed by an acceptor. The information you can get from this is that the molecules are close to each other and probably interacting with each other
If FRET occurs, the donor fluorescence decreases (quenching) and the acceptor fluorescence increases. So the ratio of acceptor/donor goes up.
When the acceptor is photobleached, donor fluorescence increases.
Cameleon is a calcium indicator based on FRET between CFP/YFP. This is one molecule consisting of CFP one end, calmodulin (calcium binding) + M13 protein sequence. When calmodulin binds calcium, the CFP and YFP are brought close together and FRET occurs. Cameleon sold by Aurora Biosciences.
| References: |
| J Biol Chem 2000 275: 17035-17042 |
| J Biol Chem 2000 275: 12769-12780 |
| J Biol Chem 2000: 8812-8816 |
| Journal of Cell Science 114, 4025-4031 (2001) |
| American Journal of Botany, 87:1547-1560 (2000) |
| Journal of Cell Science 114, 4025-4031 (2001) |
| Nature Cell Biology4, 232 - 239 (01 Mar 2002) |
| Nature Cell Biol, Vol4, Jan 2002, Pg E15 |
| J of Microscopy Vol 205, Jan 2002, pg3-14 (Peri) |
| Nat Cell Biology Vol 4, April 2002, E91 |
| http://www.cci.virginia.edu/ |
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FLIM - Fluorescence Lifetime Imaging
The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state. The process of fluorescence emission by an excited molecule occurs like radioactivity with a characteristic half-life or decay lifetime of a few nanoseconds.
The lifetime varies between different fluorescent molecules and for the same probe in a different environment. For example, the lifetimes of many fluorophores are altered by the presence of ions such as Ca 2+, Mg 2+, Cl , pH or K+. This allows the researcher to follow environmentally induced changes. FLIM does not require wavelength-ratiometric probes.
An advantage of lifetime imaging is that the absolute values of lifetimes are independent of the probe concentration, photobleaching, light scattering and the amount of excitation intensity. Fluorescence lifetime imaging (FLIM) thus offers several opportunities to study dynamic events of living cells.
Fluorescence lifetime can give information about colocalisation of two molecules (e.g.FRET).
And separation of two emissions with same spectrum.
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FCS - Fluorescence Correlation Spectroscopy
FCS measures the fluctuations of fluorescence intensity with time as molecules move in and out of a focal volume (parked beam). Small rapidly diffusing molecules produce a different pattern of fluorescence intensity than larger molecules. The time pattern (autocorrelation) can give information about the rate of diffusion of molecules and their concentration in the analysis volume.
Applications
- Diffusion
- Drug binding
- Protein folding
- Singlet/triplet kinetics
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FLIP - Fluorescence loss in photobleaching
A part of a cell at a distance from where there is an excess of fluorescent protein is bleached with a laser at low intensity. One then images the sample just before and at constant intervals after bleaching, allowing the bleached molecules to redistribute through the cell.
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FLAP - Fluorescence localization after photobleaching
The molecule to be located has 2 fluorophores, one to be bleached, and the other to act as a reference label. One can then track the distribution of the molecule after it is bleached. The FLAP signal was obtained by subtracting the bleached signal from the unbleached one, allowing the tracking of the labelled molecule.
| References: |
| J Microscopy Vol 205, Jan 2002 p 109-112 |
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FISH - Fluorescence in situ hybridization
FISH involves the preparation of short sequences of cDNA complementary to the DNA sequence of interest. These cDNA probes are labeled with fluorescent tags, and once the probes hybridize to the complementary DNA, the area of DNA fluoresces. FISH can be performed on nondividing cells as well as actively dividing cells.
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CALI - Chromophore assisted laser inactivation
CALI is a light-mediated technique used to selectively inactivate proteins within cells. This technique damages specific proteins in vivo by binding them with non-blocking antibodies conjugated with the a dye known as malachite green, that generates short lived free radicals that damage proteins.
Chromophore-assisted laser inactivation uses laser energy to inactivate specific proteins within a cell to learn more about what the functions of these proteins are
| References: |
| Nat Cell Biology Vol 4, April 2002, E91 |
| Nat Cell Biology Vol 4, April 2002, pg 1 |
| Proteomics Vol2, 2002, 241-246 |
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BRET - Bio-luminescence resonance energy transfer
Light resonance energy transfer approaches are based on the non-radiative transfer of excitation energy between the electromagnetic dipoles of an energy donor and acceptor. In the case of fluorescence resonance energy transfer (FRET), both the donor and acceptor are fluorescent molecules, whereas for bioluminescence resonance energy transfer (BRET), the donor is bioluminescent and the acceptor is fluorescent. A prerequisite for these processes is that the emission spectrum of the donor and the excitation spectrum of the acceptor must overlap and that the donor and acceptor be in close proximity.
BRET is a naturally occurring phenomenon in marine animals such as sea pansy Renilla reniformis and the jellyfish Aequorea victoria. In R. reniformis, the emitted luminescence from the degradation of coelenterazine by luciferase is transferred to green fluorescent protein (GFP), which then emits fluorescence at its characteristic wavelength.
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AFM - Atomic Force Microscopy
AFM measures the forces (at the atomic level) between a sharp probing tip attached to a cantilever spring and a sample surface.
AFM is particularly attractive to cell biologists. It can yield high resolution spatial images of live cells under near physiological conditions as well as evaluate physical parameters in biological materials, such as material properties and binding forces.
Top |  CZ CellScience Ltd.
 What is FRAP?
What is FRET?
What is FLIM?
What is FCS?
What is FLIP?
What is FLAP?
What is FISH?
What is CALI?
What is BRET?
What is AFM? |