Citation Index 3
Calibration of Fluorescence Resonance Energy Transfer
in Microscopy Using Genetically Engineered GFP Derivatives
on Nickel Chelating Beads
Douglas C. Youvan, Christopher M. Silva, Edward J. Bylina,
William J. Coleman, Michael R. Dilworth, and Mary M. Yang
Bldg. 62*
3350 Scott Blvd.
Santa Clara, CA 95054 USA
Received: 9/5/97
Accepted: 9/18/97
Ad Hoc Editor: Professor George Phillips
Abstract
Molecular reagents, software algorithms, and optics are described for calibrating an epifluorescence microscope for fluorescence resonance energy transfer (FRET) imagery. This MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra. The effectiveness of this new method relies on the use of well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. To calibrate the system, three bead types (bearing pure donor, pure acceptor, and a donor-plus-acceptor FRET mix) are imaged sequentially by three epifluorescence cubes that are fabricated to maintain proper optical image registration. A series of algorithms corrects and combines the three monochrome images into one RGB image in which FRET, acceptor, and donor fluorescence are pseudocolored red, green, and blue, respectively. The utility of this new method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by Histidine-tags. In addition, various theoretical models are considered for conformational differences in FRET pairs on the bead surface.
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© Biotechnology et alia, 1997
*Current address: 10225 Barnes Canyon Rd., A110 San Diego, CA 92121 USA