Premium
Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy
Author(s) -
Tiede LeAnn M.,
Nichols Michael G.
Publication year - 2006
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/2005-09-19-ra-689
Subject(s) - photobleaching , autofluorescence , nicotinamide adenine dinucleotide , two photon excitation microscopy , fluorescence , biophysics , chemistry , fluorescence recovery after photobleaching , laser , photochemistry , nad+ kinase , optics , biology , biochemistry , physics , enzyme
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.