In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra (hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dualwavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed

Low infra red laser light irradiation on cultured neural cells: effects on mitochondria and cell viability after oxidative stress

<p>Abstract</p> <p>Background</p> <p>Considerable interest has been aroused in recent years by the well-known notion that biological systems are sensitive to visible light. With clinical applications of visible radiation in the far-red to near-infrared region of the spectrum in mind, we explored the effect of coherent red light irradiation with extremely low energy transfer on a neural cell line derived from rat pheochromocytoma. We focused on the effect of pulsed light laser irradiation vis-à-vis two distinct biological effects: neurite elongation under NGF stimulus on laminin-collagen substrate and cell viability during oxidative stress.</p> <p>Methods</p> <p>We used a 670 nm laser, with extremely low peak power output (3 mW/cm²) and at an extremely low dose (0.45 mJ/cm²). Neurite elongation was measured over three days in culture. The effect of coherent red light irradiation on cell reaction to oxidative stress was evaluated through live-recording of mitochondria membrane potential (MMP) using JC1 vital dye and laser-confocal microscopy, in the absence (photo bleaching) and in the presence (oxidative stress) of H₂O₂, and by means of the MTT cell viability assay.</p> <p>Results</p> <p>We found that laser irradiation stimulates NGF-induced neurite elongation on a laminin-collagen coated substrate and protects PC12 cells against oxidative stress.</p> <p>Conclusion</p> <p>These data suggest that red light radiation protects the viability of cell culture in case of oxidative stress, as indicated by MMP measurement and MTT assay. It also stimulates neurite outgrowth, and this effect could also have positive implications for axonal protection.</p

Apoptosis- and necrosis-induced changes in light attenuation measured by optical coherence tomography

Optical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis had been induced. We analysed the OCT data, including both the scattering properties of the medium and the axial point spread function of the OCT system. The optical attenuation coefficient in necrotic cells decreased from 2.2 ± 0.3 mm−1 to 1.3 ± 0.6 mm−1, whereas, in the apoptotic cells, an increase to 6.4 ± 1.7 mm−1 was observed. The results from cultured cells, as presented in this study, indicate the ability of OCT to detect and differentiate between viable, apoptotic, and necrotic cells, based on their attenuation coefficient. This functional supplement to high-resolution OCT imaging can be of great clinical benefit, enabling on-line monitoring of tissues, e.g. for feedback in cancer treatment

Inhibition of cardiac contractility by 5-hydroxydecanoate and tetraphenylphosphonium ion: a possible role of mitoKATP in the response to inotropic stress.

This study investigates the role of the mitochondrial ATP-sensitive K+ channel (mitoKATP) in response to positive inotropic stress. In Langendorff-perfused rat hearts, inotropy was induced by increasing perfusate calcium to 4 mM, by adding 80 microM ouabain or 0.25 microM dobutamine. Each of these treatments resulted in a sustained increase in rate-pressure product (RPP) of approximately 60%. Inhibition of mitoKATP by perfusion of 5-hydroxydecanoate (5-HD) or tetraphenylphosphonium before induction of inotropic stress resulted in a marked attenuation of RPP. Inhibition of mitoKATP after induction of stress caused the inability of the heart to maintain a high-work state. In human atrial fibers, the increase in contractility induced by dobutamine was inhibited 60% by 5-HD. In permeabilized fibers from the Langendorff-perfused rat hearts, inhibition of mitoKATP resulted, in all cases, in an alteration of adenine nucleotide compartmentation, as reflected by a 60% decrease in the half-saturation constant for ADP [K1/2 (ADP)]. We conclude that opening of cardiac mitoKATP is essential for an appropriate response to positive inotropic stress and propose that its involvement proceeds through the prevention of stress-induced decrease in mitochondrial matrix volume. These results indicate a physiological role for mitoKATP in inotropy and, by extension, in heart failure

1H NMR, GC-EI-TOFMS, and data set correlation for fruit metabolomics: Application to spatial metabolite analysis in melon

International audienceA metabolomics approach combining 1H NMR and gas chromatography−electrospray ionization time-of-flight mass spectrometry (GC−EI-TOFMS) profiling was employed to characterize melon (Cucumis melo L.) fruit. In a first step, quantitative 1H NMR of polar extracts and principal component analyses (PCA) of the corresponding data highlighted the major metabolites in fruit flesh, including sugars, organic acids, and amino acids. In a second step, the spatial localization of metabolites was investigated using both analytical techniques. Direct 1H NMR profiling of juice or GC−EI-TOFMS profiling of tissue extracts collected from different locations in the fruit flesh provided information on advantages and drawbacks of each technique for the analysis of a sugar-rich matrix such as fruit. 1H NMR and GC−EI-TOFMS data sets were compared using independently performed PCA and multiblock hierarchical PCA (HPCA), respectively. In addition a correlation-based multiblock HPCA was used for direct comparison of both analytical data sets. These data analyses revealed several gradients of metabolites in fruit flesh which can be related with differences in metabolism and indicated the suitability of multiblock HPCA for correlation of data from two (or potentially more) metabolomics platforms