and with the machine's temperature controller also set at 25 ºC (298 K), and with accurately calibrated micropipettes. You should also check that the cuvettes are clean and dry before using them. This large initial jump in the recorded ratiometric values is due to interference with HPTS response when the transporter is added to the cuvette. This interference must be excluded from the normalisation process as this does not represent the true membrane transport process. This interference is illustrated by an HPTS transport assay conducted with a previously reported tetrapodal anion transporter, tetraphenylurea, and shows the effect of including (Figure