Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

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.

Light exposure and cell viability in fluorescence microscopy

“…Increase of phototoxicity of native cells with decreasing wavelength may be related to an increasing number of molecular species with phototoxic properties, e.g. porphyrins (Ricchelli, 1995), flavins (Hockberger et al, 1999) and possibly also nicotinamide adenine dinucleotide (NADH; Tiede & Nichols, 2006). Exogenous dyes induce further phototoxicity (depending on their concentration and triplet yield), which is most pronounced for photosensitizers, e.g.…”

Tumor cell differentiation by label-free fluorescence microscopy

“…In particular, phototoxic properties of NADH 30 and flavins 31 should be taken into account, and damage by high light exposure should be avoided. As previously reported, 17 a light dose of 25 J∕cm 2 (corresponding to 0.25 μJ∕μm 2 ) at an excitation wavelength of 375 nm or 100 J∕cm 2 (corresponding to 1 μJ∕μm 2 ) at 514 nm may be regarded as a maximum for maintaining cell viability.…”

Photodamage in deep tissue two‐photon optical biopsy of human skin

“…A second damaging effect is due to the high laser peak powers causing photoionization and plasma formation. This effect was reported to be accompanied by the onset of strong luminescence from newly formed compounds [9,12,22,23]. A third effect is of thermomechanical origin.…”