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.
"(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. "
[Show abstract][Hide abstract] ABSTRACT: This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.
Journal of Innovative Optical Health Sciences 01/2014; 7(5):1330010. DOI:10.1142/S1793545813300103 · 0.93 Impact Factor
"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 . 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. "
[Show abstract][Hide abstract] ABSTRACT: Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ′ factors exceeding 0.6 are realised for this FLIM FRET assay.
Fluorescence lifetime plate map with representative images of high and low FRET cells and corresponding dose response plot.
Journal of Biophotonics 05/2013; 6(5). DOI:10.1002/jbio.201200185 · 4.45 Impact Factor
"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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