DRBIO
FAQ's
2. What is Fluorescence Correlation Spectroscopy (FCS)?
3. What is Forster Resonance Energy Transfer?
1. What is Multiphoton Microscopy (MPM)?
Multiphoton laser spectroscopy covers a wide range of studies including photochemical reactions, molecular dynamics, micro-pharmacology and optical memory for data storage. When combined with fluorescence microscopy, non-resonant multiphoton excitation (MPE) provides 3D-submicron imaging resolution of fluorescent markers (extrinsic or intrinsic) deep in living biological tissues. The localization of multiphoton excitation minimizes the overall photodamage to the specimen. MPM apparatus.
Selected Webb publications on MPM:
Denk, W., J. H. Strickler and W. W. Webb, "Two-Photon Laser Scanning Fluorescence Microscopy," Science 248, 73-76, 1990
Xu, C., W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy," PNAS 93(20), 10763-10768, 1996
Williams, R. M., W. R. Zipfel and W. W. Webb, "Multiphoton microscopy in biological research," Current Opinion in Chemical Biology 5, 603-608, 2001
Zipfel, W. R., R. M. Williams and W. W. Webb, "Nonlinear Magic: Multiphoton Microscopy in the Biosciences," Nature Biotechnology 21(11), 1369-1377, 2003
2. What is Fluorescence Correlation Spectroscopy (FCS)?
"Fluorescence correlation spectroscopy examines chemical and photophysical dynamics of dilute molecular solutions by measurement of the dynamic optical fluctuations of the fluorescence of a few molecules, even averaging less than one molecule at a time, in open focal volumes that are usually less than a femtoliter (<10-18 m3). It applies the same principles of statistical thermodynamics as does quasi-elastic light scattering. Molecular interactions, conformational changes, chemical reactions, and photophysical dynamics that are not ordinarily detectable by quasi-elastic light scattering can be analyzed by fluorescence correlation spectroscopy in cases in which molecular fluorescence changes in the dynamic range 10-7–102 s." © 2001 Optical Society of America. From Webb, W.W., "Fluorescence Correlation Spectroscopy: Inception, biophysical experimentations and prospectus," Applied Optics 40(24), 3969-3983, 2001.
3. What is Förster Resonance Energy Transfer (FRET)?
This technique is also know as fluorescence resonance energy transfer. FRET is a quantum mechanical process in which the radiationless transfer of excitation energy occurs between a donor blue fluorescent protein (BFP) fluorophore and an acceptor green fluorescent protein (GFP) fluorophore. Efficiency of this energy transfer varies inversely with the sixth power of the distance between the fluorophore-tagged proteins. Resonance energy transfer can occur only at 3ž4 ~100 Å, and the efficiency of energy transfer is very sensitive to the distance separating BFP- and GFP-fusion proteins.
Last
update:
February 10, 2005