Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH

Fluorescentdeterminations of NADH in porcine heart mitochondria were subject tosignificant errors caused by alterations in inner filter effects duringnumerous metabolic perturbations. These inner filter effects wereprimarily associated with changes in mitochondrial volume andaccompanying light scattering. The observed effects were detected in astandard commercial fluorometer with emission orthogonal to theexcitation light path and, to a lesser extent, in a light path geometrydetecting only the surface fluorescence. A method was developed todetect and correct for inner filter effects on mitochondrial NADHfluorescence measurements that were independent of the optical pathgeometry using an internal fluorescent standard and linearleast-squares spectral analysis. A simple linear correction with theinner fluorescence reference was found to adequately correct for innerfilter effects. This approach may be useful for other fluorescenceprobes in isolated mitochondria or other light-scattering media.