ion one is trying to measure. Other ions cause interference and may combine with thetrue signal thus making it difficult to assess the exact change in activity of the ion onewishes to measure. In practice what this does is to set a detection limit for theelectrode. Generally, ﬂuorescent dyes are selective for the ion in question, but theycan suffer from direct interference or changes in autoﬂuorescence produced by, forexample, pharmacological compounds.
. This is relatively straightforward for ion-sensitive electrodes, whichcan be calibrated separately, but can be difficult with ﬂuorescent dyes.
. The sensitivity of ion-sensitive microelectrodes toelectrical interference requires that they be well shielded. Fluorescent dyes obviouslydo not suffer from such problems but they must be used in the dark which can also putconstraints on the design of the experimental area.
. Ion-sensitive microelectrodes themselves are inert measuring devices inthat they do not add or remove anything from the cell under test. They do, however,require impalement of the cell membrane. Fluorescent dyes are not inert in the sameway. Ca
indicators, for example, may signifcantly increase the Ca
bufferingpower of the cell.
2. Ion-sensitive electrode measurements
Ion activity and ion concentration
If we were to grind up a biological preparation and assess the calcium content of thetissue by atomic absorption spectrophotometry and then compare this value with oneproduced by an ion-sensitive microelectrode or ﬂuorescent indicator we would ﬁndthat the totalcalcium concentration would be of the order of millimoles per litrewhilst the freeintracellular calcium ion concentration would be 100 or so nanomolesper litre. Thus a huge percentage of the calcium is bound to moieties within the celle.g. intracellular organelles and buffers. The differentiation between free ionconcentration and total ion concentration is a simple matter. However, what is thedifference between free ion concentration and activity? As another illustrationconsider a solution of 150 mM NaCl in water. The NaCl would fully ionise so theconcentration of Na
]) in the solution would be 150 mM. However, themutual electrostatic repulsion between the similarly charged species and attractionbetween anions and cations reduces their mobility and freedom especially as theconcentration of the solution becomes higher. In other words, the ions exhibit non-ideal behaviour, which is not due to incomplete ionization but to the existance of inter-ionic forces. Thus the activity of the Na
ions in solution or their effectiveconcentration is less than their total concentration. Only when the solute is inﬁnitelydiluted will its concentration equal its activity. It is the activity of the ion which isused in calculations related to thermodynamic processes. The constant that relates theion’s activity to concentration is the activity coefficient (
) such that: