Phycobiliprotein-Fab conjugates as probes for single particle fluorescenceimaging

Background and Methods: Single particle fluorescence imaging (SPFI) is a re cently developed method that has provided a powerful approach to observing receptor movement and associations at high spatial resolution. It provides a noninvasive alternative to the existing bio-chemical techniques. It can a lso quantify and resolve molecular interactions at the cell surface at a na nometer scale. Probes that have been used in the past to study mobility and associations of cell surface receptors have many limitations. These includ e concerns about the specificity of the probes, the possibility that their size interferes with the receptor once hound to it, the nonuniform fluoresc ence, and the questionable stoichiometry. Results: In this study, we have generated phycobiliprotein-Fab conjugates, and have shown that they are a significant advance on existing probes for S PFI studies. They are small in size, highly specific, highly fluorescent, o f known stoichiometry, photostable, emit uniform fluorescence, and are gene rally well defined. Conclusions: It is highly important that when studying receptor mobility or associations, fully characterized probes are used. Phycoerythrin(PE)-Fab p robes provide us with the perfect tool for SPFI, and a system with a wide r ange of applicability to study any cell surface receptor against which a mo noclonal antibody exists. (C) 2000 Wiley-Liss, Inc.