Microfluorometric study of oxygen dependence of (1"-pyrene butyl)-2-rhodamine ester probe in mitochondria of living cells

The access to oxygen concentration is of importance in various organelles of living cells, especially in mitochondria. A new probe, (1"-pyrene butyl)-2-rhodamine ester, was designed to target this organelle. We present here the properties of the probe in such an environment. Microspectrofluorometry confirms the localization of the probe in the mitochondrial environment at low doses (1 microM). At these doses, the cell toxicity experiments show no effect on the cell growth. The emission spectrum measured at an excitation wavelength of 340 nm (pyrene centered) indicates energy transfer from the pyrene to the rhodamine chromophore, as also observed in an ethanol solution. With excitation at 337 nm, the excited state decays biexponentially with lifetime decays of 6-9 ns and 90 ns. The first corresponds to the intrinsic fluorescence of the cell and the latter corresponds to the pyrene chromophore. In degassed conditions the pyrene lifetime decay increases up to 130 ns. Under an oxygen atmosphere the lifetime decays decrease to 62 ns. The lifetime changes with the oxygen concentration were compared with the range obtained during our previous study in ethanol solution (5-220 ns). The observed differences were interpreted by studying the lifetime of the probe in simplified environments, liposome suspensions and protein solutions. In this paper we show that the new probe can be used to measure the fluctuation of oxygen concentration in the surroundings of mitochondria.     

Determination of metallic contaminants on Ge wafers using direct- and droplet sandwich etch-total reflection X-ray fluorescence spectrometry

An analysis methodology for the metallic contamination control of Ge wafer substrates has been developed and evaluated for six elements (K, Ca, Cr, Fe, Ni and Zn). Detection limits (DL) of Direct-total reflection X-ray fluorescence spectrometry (D-TXRF) analysis on Ge wafers have been determined and found to be at the E10 at/cm² level. The values have been found to be a factor between 1 and 3 higher than on Si wafers, exclusively caused by differences in the background intensity. Additionally, a preconcentration procedure based on the Droplet sandwich etch (DSE) method has been developed. This method relies on the transfer of the surface and subsurface contaminants from the wafer to the liquid phase by wet chemical etching. Application of the DSE method on reference Ge wafers followed by analysis of the etch liquid by TXRF resulted in recovery rates (RR) of 40%. In an optimization study, it was found that the main DSE method parameters had limited influence on the RR. However, a detection efficiency study clearly demonstrated an underestimation by the TXRF analysis. An independent analysis for Ca, Cr, Fe and Zn by GF-AAS resulted in RR varying at approximately 100%. By internal standardization with the element La for the TXRF analysis, recovery rates could be increased to the 60% level. This underestimation by TXRF may find an origin in a matrix effect caused by the Ge etch products. By application of the developed DSE-TXRF method, DL at the E9 at/cm² level could be realized, with values, which are at least one order of magnitude lower compared to the DL of D-TXRF on Ge wafers.     

Grazing incidence-X-ray fluorescence spectrometry for the compositional analysis of nanometer-thin high-kappa dielectric HfO2 layers.

In future microelectronic devices, SiO2 as a gate dielectric material will be replaced by materials with a higher dielectric constant. One such candidate material is HfO2. Thin layers are typically deposited from ligand-containing precursors in chemical vapor deposition (CVD) processes. In the atomic layer deposition (ALD) of HfO2, these precursors are often HfCl4 and H2O. Obviously, the material properties of the deposited films will be affected by residual ligands from the precursors. In this paper, we evaluate the use of grazing incidence--and total reflection-X-ray fluorescence spectrometry (GI-XRF and TXRF) for Cl trace analysis in nanometer-thin HfO2 films deposited using ALD. First, the results from different X-ray analysis approaches for the determination of Hf coverage are compared with the results from Rutherford backscattering spectrometry (RBS). Next, we discuss the selection of an appropriate X-ray excitation source for the analysis of traces within the high-kappa: layers. Finally, we combine both in a study on the accuracy of Cl determinations in HfO2 layers.     

Determination of pesticide residues in lettuce by gas chromatography with electron-capture detection

A sensitive, rapid, and simple multiresidue method for the simultaneous determination of 19 pesticides in different varieties of lettuce (Lactuca sativum) was developed. Lettuce samples were extracted by homogenization with acetone and partitioned into ethyl acetate-cyclohexane. Subsequent sample cleanup was not needed. Final determination was made by capillary gas chromatography (GC) with electron-capture detection (ECD). Confirmation analysis of pesticides was performed by GC coupled with mass spectrometry in the selected ion monitoring mode. The average recovery by the GC-ECD method obtained for these compounds varied from 66.4 to 119.2% with relative standard deviations < 7.7%. The GC-ECD method has good linearity, and the detection limit for the pesticides studied varied from 0.1 to 3.8 microg/kg. The proposed method was used to determine pesticide levels in different types of lettuce grown in soils from experimental fields.     

Lifetime of fluorescent pyrene butyric acid probe in single living cells for measurement of oxygen fluctuation

We study the fluorescence lifetime of the well-known 1-pyrene butyric acid (PBA) to assess oxygen concentrations in living cells. The behavior of the probe is first studied in water, ethanol, protein solution and liposome suspension. The Stern-Volmer plot of these solutions is linear, and the bimolecular reaction rate constant agrees with previous observations. In single living cells, the PBA lifetime decreases with oxygen concentration (185 to 55 ns). The probe lifetime differences between living cells and liposome suspension, especially under nitrogen atmosphere, suggest a supplemental pathway for the deactivation of the probe. We simplify further the complex living cells system by stopping the cell functions and studying freshly fixed cells. In this case, we obtained an increase of PBA lifetime under nitrogen atmosphere (215 ns).     

Role of Nuclear Factor-KB in Colon Cancer Cell Apoptosis Mediated by Aminopyropheophorbide Photosensitization

Aminopyropheophorbide (APP) is a second generation of photosensitizer for photodynamic therapy (PDT). We demonstrated that APP strongly absorbed red light and, after being taken up by colon cancer cells (HCT-116 cells), was localized in cytoplasmic and internal membranes but not in mitochondria. The APP-mediated photosensitization was cytotoxic for HCT-116 cells through an induction of apoptosis. Indeed, DNA fragmentation (DNA laddering and terminal deoxyuridine nick-end labeling) and chromatin condensation (4',6-diamidine-2'-phenylindole staining) could be visualized soon after photosensitization. Because nuclear factor (NF)-kappa B is involved in the response to many photosensitizers, we also demonstrated its nuclear translocation in two waves: a rapid and transient one, followed by a slow and sustained phase. The NF-kappa B turned out to be involved in an antiapoptotic response to APP-mediated photosensitization because the HCT-116 cell line expressing the dominant negative mutant of inhibitor-kappa B alpha was more sensitive to apoptosis as measured by DNA fragmentation and caspase activation. These data unambiguously show that a membrane-located photosensitizer can lead to effective apoptosis, reinforcing the idea that PDT can be an effective means to eradicate colon cancer cells.