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Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level

Paper ID Volume ID Publish Year Pages File Format Full-Text
31116 44547 2009 12 PDF Available
Title
Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level
Abstract

Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ∼1.8-fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.

Keywords
NADH; Hs578T; Hs578Bst; Two-photon FLIM; Associated anisotropy; Dehydrogenase
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Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Photochemistry and Photobiology B: Biology - Volume 95, Issue 1, 2 April 2009, Pages 46–57
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us