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

Stephanie A. French, Paul R. Territo, Robert S. Balaban

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Fluorescent determinations of NADH in porcine heart mitochondria were subject to significant errors caused by alterations in inner filter effects during numerous metabolic perturbations. These inner filter effects were primarily associated with changes in mitochondrial volume and accompanying light scattering. The observed effects were detected in a standard commercial fluorometer with emission orthogonal to the excitation light path and, to a lesser extent, in a light path geometry detecting only the surface fluorescence. A method was developed to detect and correct for inner filter effects on mitochondrial NADH fluorescence measurements that were independent of the optical path geometry using an internal fluorescent standard and linear least-squares spectral analysis. A simple linear correction with the inner fluorescence reference was found to adequately correct for inner filter effects. This approach may be useful for other fluorescence probes in isolated mitochondria or other light-scattering media.

Original languageEnglish (US)
Pages (from-to)C900-C909
JournalAmerican Journal of Physiology - Cell Physiology
Volume275
Issue number3 44-3
StatePublished - Oct 5 1998

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Keywords

  • Calcium
  • Heart
  • Light scattering
  • Linear least squares
  • Mitochondria
  • Optical methods
  • Pig

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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