Abstract
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 language | English |
|---|---|
| Pages (from-to) | 656-664 |
| Number of pages | 9 |
| Journal | Photochemistry and Photobiology |
| Volume | 82 |
| Issue number | 3 |
| DOIs | |
| State | Published - May 2006 |
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All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biophysics
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Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy. / Tiede, LeAnn M.; Nichols, Michael G.
In: Photochemistry and Photobiology, Vol. 82, No. 3, 05.2006, p. 656-664.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy
AU - Tiede, LeAnn M.
AU - Nichols, Michael G.
PY - 2006/5
Y1 - 2006/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33745805869&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33745805869&partnerID=8YFLogxK
U2 - 10.1562/2005-09-19-RA-689
DO - 10.1562/2005-09-19-RA-689
M3 - Article
C2 - 16426080
AN - SCOPUS:33745805869
VL - 82
SP - 656
EP - 664
JO - Photochemistry and Photobiology
JF - Photochemistry and Photobiology
SN - 0031-8655
IS - 3
ER -