In Receptor sensitivity in bacterial chemotaxis by Victor Sourjik and others, Victor Sourjik and Howard C. Berg used a cyan fluorescent tag on CheZ as FRET donor and a yellow tag on CheY as FRET acceptor. Escherichia coli biological cells adhered to a polylysine-coated coverslip in a continuously perfused chamber. A fluorescence microscope excited them with a -nanometre laser; split emission bands fed two photon-counting detectors. The yellow/cyan emission ratio fell when attractant was added and rose when it was removed.[ref]
Receptor sensitivity in bacterial chemotaxis by Victor Sourjik and others. Introduction, Materials and Methods: Preparation of Cells and Data Acquisition; Results and Figure 1
Saturating attractant increased cyan emission by an amount matching selective bleaching of the yellow tag: removing the FRET acceptor supported relief of FRET donor transfer. Strains carrying the tagged pair but lacking other chemotaxis machinery showed no stimulus-dependent emission ratio change. Weak illumination reduced bleaching drift.[ref]
Receptor sensitivity in bacterial chemotaxis by Victor Sourjik and others. Data Analysis and Results: acceptor bleaching comparison, low illumination, and flhC negative control
The inference is a change in the population of nearby tagged CheY/CheZ pairs associated with CheY phosphorylation. It neither measures each pair's separation nor directly observes motor motion. The readout also averages many biological cells, and perfusion takes finite time. The pathway interpretation belongs to the chemotaxis FRET assay; the general measurement lesson is that molecular association can alter FRET while the geometry of an individual associated pair stays the same.
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