Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress

Institute of Anatomy II, Friedrich Schiller University, Jena, Germany.
Journal of Microscopy (Impact Factor: 2.33). 10/1996; 183(Pt 3):197-204. DOI: 10.1046/j.1365-2818.1996.910650.x
Source: PubMed

ABSTRACT By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UVA radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser, focused through high-numerical-aperture microscope optics, provided diffraction-limited microbeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster ovary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm-2) resulted in oxidative stress correlated with increased autofluorescence intensity, onset of nuclear fluorescence, and a fluorescence lifetime decrease. The cellular response to femtosecond NIR microbeams depended significantly on peak power. Peak powers above a threshold value of about 0.5 kW (average power: 6 mW). 0.55 kW (7 mW) and 0.8 kW (10 mW) at 730 nm, 760 nm and 800 nm, respectively, resulted in the onset of short-lived luminescence with higher intensity (100 x) than the intracellular NAD(P)H fluorescence. This luminescence, accompanied by destruction of cellular morphology, was localized and occurred in the mitochondrial region. In contrast, beams at a power of less than 0.5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.

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    • "(b) Photodamage may also be caused by mechanisms resulting from the high peak power of the fem to second laser pulses. There are indications that dielectric breakdown occasionally occurs.30 (c) Most importantly, one-and two-photon absorption of high-power infrared radiation may also produce thermal damage. "
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    Journal of Innovative Optical Health Sciences 01/2014; 7(5):1330010. DOI:10.1142/S1793545813300103 · 0.93 Impact Factor
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    • "In spite of these advantages, FLIM has not yet made a significant impact on drug discovery, partly due to a lack of available FLIM instrumentation for automated multiwell plate readouts. FLIM is often implemented using laser scanning microscopes with time correlated single photon counting (TCSPC) [22–24], for which the sequential pixel acquisition of this approach typically results in data acquisition times of 10's–100's of seconds per cell to acquire sufficient detected photons, depending on the sample brightness, with the accuracy of fluorescence lifetime determination being proportional to the square root of the number of photons detected [25]. Such acquisition times are impractical for HCA and, if the excitation intensity is increased to permit much faster imaging, there are significant issues with photobleaching and phototoxicity. "
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    Journal of Biophotonics 05/2013; 6(5). DOI:10.1002/jbio.201200185 · 4.45 Impact Factor
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    • "Chu et al. then showed a multimodal imaging including TPEF1SHG1THG in 2001 (Chu et al., 2001). FLIM was observed in laser scanning system in 1989 (Bugiel et al., 1989) and combined with TPFE in 1996 (König et al., 1996; Yu et al., 1996). Since then, it became clear that FLIM is a technique that can be integrated with all different NLO microscopies providing important information about the molecules chemical environment (Zoumi et al., 2002). "
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