Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress
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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- "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). "
ABSTRACT: In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.Microscopy Research and Technique 10/2012; 75(10):1383-94. DOI:10.1002/jemt.22078 · 1.17 Impact Factor
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- "In contrast , cell autofluorescence was fitted best to a threeexponential model with longer mean lifetime (AEsae = 7.19 vs. 0.47 ns, cell vs. free NADH). Others have found a similar complex decay for cell autofluorescence     . The longer lifetimes may reflect protein binding of NAD(P)H: for example, Jameson et al.  found that NADH bound to mitochondrial malate dehydrogenase extended the lifetime from 2 to 10 ns. "
ABSTRACT: The aim of this study was to test the hypothesis that glucose can be monitored non-invasively by measuring NAD(P)H-related fluorescence lifetime of cells in an in vitro cell culture model. Autofluorescence decay functions were measured in 3T3-L1 adipocytes by time-correlated single-photon counting (excitation 370nm, emission 420-480nm). Free NADH had a two-exponential decay but cell autofluorescence fitted best to a three-exponential decay. Addition of 30mM glucose caused a 29% increase in autofluorescence intensity, a significantly shortened mean lifetime (from 7.23 to 6.73ns), and an increase in the relative amplitude and fractional intensity of the short-lifetime component at the expense of the two longer-lifetime components. Similar effects were seen with rotenone, an agent that maximizes mitochondrial NADH. 3T3-L1 fibroblasts stained with the fluorescent mitochondrial marker, rhodamine 123 showed a 16% quenching of fluorescence intensity when exposed to 30mM glucose, and an increase in the relative amplitude and fractional intensity of the short lifetime at the expense of the longer lifetime component. We conclude that, though the effect size is relatively small, glucose can be measured non-invasively in cells by monitoring changes in the lifetimes of cell autofluorescence or of a dye marker of mitochondrial metabolism. Further investigation and development of fluorescence intensity and lifetime sensing is therefore indicated for possible non-invasive metabolic monitoring in human diabetes.Journal of Photochemistry and Photobiology B Biology 09/2005; 80(2):122-9. DOI:10.1016/j.jphotobiol.2005.04.001 · 2.80 Impact Factor
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- "Hence photodamage to the sample is minimized as is energy transfer to the sample (Denk et al 1990). This makes two-photon microscopy suitable for non-invasive imaging both in vitro and in vivo thus extending the range of samples that we are able to investigate from gels, to excised tissue, to cell culture and finally to living tissue (Konig et al 1996). Of particular relevance to this study is the early work by Masters et al (1997) using two-photon excitation to image skin in vivo and later work by Hendriks and Lucassen (2000) and Masters and So (2001) using twophoton excited fluorescence and single photon reflectance to image skin in vivo. "
ABSTRACT: This study investigates the optical effects observed from uncoated and protein vaccine coated gold microparticles while imaging with two-photon excitation in the Mie scattering regime. When observed with time correlated single photon counting fluorescence lifetime microscopy, the emission from the gold microparticles appeared as an intense instrument-limited temporal response. The intensity of the emission showed a second-order dependence on the laser power and frequency doubling of the emitted light was observed for fundamental light between 890 and 970 nm. The optical effect was attributed to two-photon induced second harmonic generation. The vaccine coated gold microparticles had a much weaker second harmonic signal than the uncoated gold microparticles. Chemical analysis of the surface of the gold microparticles revealed that the vaccine coating decreases the surface charge thereby diminishing the observed second harmonic signal. These optical properties can be exploited to identify both the location of the protein vaccine coating as well as the gold microparticles in vitro and potentially to investigate the vaccine delivery kinetics in vivo.Physics in Medicine and Biology 09/2004; 49(16):3603-12. DOI:10.1088/0031-9155/49/16/008 · 2.92 Impact Factor