Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy

LeAnn M. Tiede, Michael G. Nichols

Research output: Contribution to journalArticle

25 Scopus citations


Endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides an intrinsic indicator of the cellular metabolic state, but prolonged monitoring is limited by photobleaching and/or phototoxicity. Multiphoton excitation of NADH by ultrashort, 740-nm laser pulses provides a significant improvement over UV excitation by eliminating peripheral photobleaching; however, molecules within the subfemtoliter excitation volume remain susceptible. We have investigated the photophysical mechanisms responsible for multiphoton photobleaching of NADH in living cells to permit the imaging technique to be optimized. The loss of fluorescence because of multiphoton photobleaching was measured by repetitively imaging individual planes within rat basophilic leukemia cells. The photobleaching rate was proportional to the fourth power of the laser intensity. Based on these measurements, we propose a double-biphotonic, four-photon photobleaching mechanism and estimate the quantum yield of photobleaching of intracellular NADH to be 0.0073 ± 0.0002 by this mechanism. In addition to photobleaching, the development of bright, punctate fluorescent lesions can also be observed. The frequency of lesion formation also increased approximately as the fourth power of the laser intensity after an intensity-dependent threshold number of images had been exceeded. The consequences for two-photon metabolic imaging are discussed.

Original languageEnglish
Pages (from-to)656-664
Number of pages9
JournalPhotochemistry and Photobiology
Issue number3
Publication statusPublished - May 2006


All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics

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