Cell surface G-protein-coupled receptors are notoriously difficult to localise accurately, even in fixed tissue using antibodies, due to the non-specificity caused by the high degree of conservation of sequence, or using radioligands due to the inherent low resolution of autoradiography. However, high affinity fluorescent ligands based on "antagonist" drugs/ligands could be used in a manner analogous to radioligands, but with much higher spatial resolution and in real-time on live tissue, if their concentration can be measured photometrically.

 
The structure above is a fluorescent alpha1-adrenoceptor antagonist based on prazosin. This particular form of BODIPY is excited at 488nm and emits above 515nm. The compound was obtained from Molecular Probes and is listed in their catalogue as "BODIPY FL-prazosin" but since it lacks the furan group which defines prazosin, as opposed to other compounds which share the quinazolinyl piperazine group, such as doxazosin, we refer to it by an acronym, "QAPB", derived from its chemical name (quinazolinyl piperazine borate-dipyrromethene).
QAP-B-induced fluorescence is shown on two similar cells, one in the absence (a) , the other in the presence (b), of the alpha1-adrenoceptor agonist phenylephrine (10µM). This view shows the overall reduction in fluorescence produced by phenylephrine. Measuring the "total" fluorescence, the cell exposed to phenylephrine had 30% of that on the other cell. This difference is likely to be due to competition between the two ligands rather than down regulation of receptors since the time of exposure was short (20 min). c. The sub-cellular location of QAP-B (30nM) binding is shown. (ci) shows a single optical section (x-y plane) through the middle of the cell. The bright areas indicate regions of intense binding at the cell edges. An impression of the three dimensional distribution can be obtained by re-sectioning the cell. y-z (cii) and x-z (ciii) sections are shown: these are taken from between the vertical or horizontal lines, respectively. These views confirm that the majority of intense staining is confined to the cell membrane, although some light staining can be seen inside the cell.
Images a-c show 3D localisation of QAPB-associated fluorescence (100nM) on the cell membrane (yellow) and cytosol (blue) of rat-1-fibroblasts transfected with alpha1a-adrenoceptors. 3D images were constructed using the ISO-surface module of IMARIS on a SGI workstation. Two ISO values were selected, one for the surface (s) and another for the intracellular (i) QAPB signal. A separate ISO value was selected for the nucleus (red). Images b and c are cut to show the location of intracellular QAPB-associated fluorescence.