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Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH
S A French1, P R Territo, R S Balaban
1Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892-1061, USA.
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.
Insights
Inner filter effects can cause errors in mitochondrial NADH fluorescence measurements. A new method using an internal standard corrects these errors, improving accuracy for mitochondrial studies.
Area of Science:
- Biochemistry
- Cell Biology
- Analytical Chemistry
Background:
- Fluorescence measurements of NADH in mitochondria are crucial for studying cellular respiration.
- Metabolic changes can alter mitochondrial volume and light scattering, leading to inner filter effects.
- These effects introduce significant errors in NADH fluorescence quantification.
Purpose of the Study:
- To develop a method for detecting and correcting inner filter effects in mitochondrial NADH fluorescence.
- To ensure accurate fluorescence measurements independent of optical path geometry.
- To provide a reliable approach for studying mitochondrial function.
Main Methods:
- Utilized a standard commercial fluorometer with different optical path geometries.
- Employed an internal fluorescent standard for correction.
- Applied linear least-squares spectral analysis to correct for inner filter effects.
- Validated a simple linear correction method.
Main Results:
- Inner filter effects, caused by mitochondrial volume changes and light scattering, significantly impacted NADH fluorescence.
- The developed method successfully detected and corrected for these effects.
- A simple linear correction using an internal reference proved adequate.
- The correction method was independent of the fluorometer's optical path geometry.
Conclusions:
- A robust method was established to correct for inner filter effects in mitochondrial NADH fluorescence.
- This approach enhances the accuracy of fluorescence-based mitochondrial studies.
- The technique may be applicable to other fluorescent probes in light-scattering biological samples.