Confocal patch-clamp fluorometry (PCF) is a method combining patch-clamp techniques and confocal fluorescence microscopy for simultaneous investigation of channel activation and ligand binding in ion channels. The method has been used successfully to study CNGA2 olfactory channels and HCN2 pacemaker channels. The fluorescent ligands fcAMP and fcGMP, and the reference dye DY647, used in these studies are negatively charged. Their fluorescence signals in the volume between patch membrane and pipette tip can depend significantly on the applied voltage if the conditions are not optimal, indicating a difference between the actual ligand concentration at the patch and in the bath.

Using confocal PCF and fluorescence correlation spectroscopy, we analyzed this phenomenon and looked for conditions to minimize it. (1) When investigating voltage independent or only slightly voltage dependent ion channels, we demonstrate the benefit of using low voltages. (2) When investigating ion channels with high single channel conductance and large open probability, we show that a reduced expression is helpful to keep the membrane resistance reasonably high. (3) In general, we recommend the use of pipette tips with a planar surface following the fabrication protocol of Hilgemann (1995). These pipette shapes avoid most of the voltage drop across the tip and thus most of the described concentration errors.

It is concluded that in inside-out macropatches with large currents the concentration of charged molecules at the internal face of the patch can significantly deviate from that in the bath solution, generating relevant errors. Using appropriate experimental conditions can largely avoid the problem.