Measurement of the time of flight of photons into the skin: influence of site, age and gender, correlation with other skin parameters.

Background/purpose: The speed of light (time of flight) into the skin is obviously relied to its structure, and might appear as a tool for non-invasive investigation of skin physico-chemical properties, among them aging is of primary importance. Though already published, such time of flight measurements have never been extensively correlated with other well-documented skin parameters such as localization, the influence of gender and age, the elasticity and roughness, and the water trans-epidermal diffusion (TEWL).

Methods: A specific practical device was designed to routinely measure the time of flight (TOF) of the light into the human skin 'in vivo', in a totally non-invasive process.

New generation photon-counting cameras: algol and CPNG.

Algol and Comptage de Photons Nouvelle Génération (CPNG) are new generation photon counting cameras developed for high angular resolution in the visible by means of optical aperture synthesis and speckle interferometry and for photon noise limited fast imaging of biological targets. They are intensified CCDs. They have been built to benefit from improvements in photonic commercial components, sensitivity, and personal computer workstations processing power.

Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials.

The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering.

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction.

Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa.

Analysis of the fluorescence of monodansylcadaverine-positive cytoplasmic structures during 7-ketocholesterol-induced cell death.

Objective: To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS).

Study Design: After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells.

Analysis of the distribution of MRI contrast agents in the livers of small animals by means of complementary microscopies.

Background: Magnetic resonance imaging (MRI) contrast agents contain magnetic molecules such as iron (Fe) or gadolinium (Gd) that are injected in vivo into rats or mice to study their distribution inside the liver. Fluorescent europium (Eu) can be used as a model of Gd to obtain comparable information of this distribution of corresponding contrast agents. In a similar approach, Fe can be attached to Texas Red and used as a model of ferumoxides and be detected by fluorescence.

Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342.

Objective: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS).

Study Design: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu.

Intracellular Mg2+ diffusion within isolated rat skeletal muscle fibers.

Intracellular free magnesium concentration ([Mg2+]i) was measured in enzymatically isolated rat skeletal muscle fibers using the fluorescent dye mag-indo-1. The change in [Mg2+]i produced by a local intracellular microinjection of magnesium pidolate (magnesium pyrrolidone carboxylate) was measured at a given distance from the injection site. In one series of experiments this protocol was tested on isolated fibers that were completely embedded into silicone grease: under these conditions, the injection produced an increase in [Mg2+]i that reached a steady level some time following the injection.

Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells.

Objective: To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS).

Study Design: Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope.

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations.

Background: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments).

Methods: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope.

Firefly luciferase generates two low-molecular-weight light-emitting species.

A bioluminescent D-luciferin-luciferase mixture is separated by gel filtration during the time course of the reaction. A simultaneous analysis with an UV-visible diode array detector and an on-line luminometer gives nonsuperimposable chromatograms. Luminescence recordings display three peaks, one associated with the enzyme (light-emitting species 1: LES(1)), and two other species free from the luciferase: LES(2), with a luciferyl-adenylate-like spectrum and LES(3).

Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra.

A laboratory-made spectroluminometer was used to analyse the light emitted by firefly (Photinus pyralis) luciferase reacting with several nucleotide derivatives. The analysis of the light emission in the presence of ATP or dATP provides some evidence that the enzyme has two nucleotide binding sites, each one leading to the formation of a complex emitting mainly at 575 nm (ATP) or 610 nm (dATP). AMP is able to displace dATP from the second site (610 nm) to the first one.

Fourier analysis of electron micrographs of positively stained collagen fibrils: application to type I and II collagen typing.

Type I and II collagen (native-type) fibrils, positively stained with uranyl acetate, present typical periodic (D = 67 nm) cross-striation patterns. Although the two patterns are similar, the distributions of charged amino acids along the type I and II collagen molecules are different. After optical diffraction analysis or computer image processing of electron micrographs, different Fourier transforms were obtained from type I and II collagen fibrils, either as native fibrils or after in vitro reconstitution from purified molecules.

Measurements of intracellular Mg2+ concentration in mouse skeletal muscle fibers with the fluorescent indicator mag-indo-1.

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured.

A microspectrophotometric study on the physicochemical properties of antipyrylazo III injected into rat myoballs for measuring free magnesium ion.

The accurate measurement of the intracellular concentration of free magnesium ions ([Mg2+]i) is essential for evaluating the role of Mg2+ in cellular functions. Among the specific (compared to fluorescent indicators) metallochromic dyes, antipyrylazo III (APIII) appears to be most suitable for measuring such concentrations in vertebrate cells according to in vitro studies. In this work, the intracellular physicochemical properties of APIII as a Mg2+ indicator were investigated in the cultured rat myoball by means of a microspectrophotometric technique, in order to obtain an accurate measurement of [Mg2+]i.

Intracellular Ca2+ changes and Ca2+-activated K+ channel activation induced by acetylcholine at the endplate of mouse skeletal muscle fibres.

1. Enzymatically isolated skeletal muscle fibres were used to investigate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+]i) measured using the indicator indo-1 and single channel activity using the patch clamp technique. 2.

Measurements of sarcomere dynamics simultaneously with auxotonic force in isolated cardiac cells.

We developed an easy to use and non-invasive method to study sarcomere motion of enzymatically isolated myocytes which can be simultaneously combined with auxotonic force detection, thus being very useful when studying the contractile performance of cardiac cells. This method basically consists in analyzing the periodicity of the cell striation pattern using the Cooley-Tukey fast Fourier transform (FFT) algorithm on a video image of the cell during the course of the experiment. A longitudinal fraction of the cell image is recorded with a CCD TV camera, digitized, then transiently stored on a computer and used to calculate the spectrum corresponding to the distribution of the sarcomere lengths (SL).

Flexibility of myosin in pyrophosphate and NaCl solutions. An electric birefringence study.

The orientational relaxation time of myosin has been reported as 38 microseconds when measured in pyrophosphate media at elevated pH (Hvidt et al. 1984) and as 17 microseconds when measured in 0.3 M KCl at pH 7.

Microinjection of a conserved peptide sequence of p34cdc2 induces a Ca2+ transient in oocytes.

The product of the yeast cell cycle control gene cdc2, and its homologs in higher eukaryotes (p34cdc2), all contain a perfectly conserved sequence of 16 amino acids that has not been found in any other protein sequence. Microinjection of this peptide triggers a specific increase in the concentration of intracellular free Ca2+ that originates from intracellular stores in both starfish and Xenopus oocytes. Thus, p34cdc2 might interact through its conserved peptide domain with some component of the Ca2(+)-regulatory system.

Electric birefringence study of rabbit skeletal myosin subfragments HMM, LMM, and rod in solution.

Electric birefringence measurements and depolarized light scattering experiments were performed with HMM, LMM, and rod, the three fragments of myosin, under conditions (0.3 M KCl, 0.02 M PO4, pH 7.

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study.

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA.

A hydrodynamic study of collagen fibrillogenesis by electric birefringence and quasielastic light scattering.

Neutral soluble collagen was extracted from lathyritic rat skin under proteolysis-inhibited conditions. Purified solutions were characterized by electric birefringence and heterodyne beat quasi-elastic light-scattering techniques under conditions where the monomeric form was stable (at 4 degrees C in 0.032 M phosphate buffer at pH 7.

Conformation of chromatin oligomers. A new argument for a change with the hexanucleosome.

Quasielastic laser light scattering measurements have been made on chromatin oligomers to obtain information on the transition in their electrooptical properties, previously observed for the hexameric structures [Marion, C. and Roux, B. (1978) Nucleic Acids Res.