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. This system was tested on volunteers, to relate the TOF parameter to the widely investigated skin properties already mentioned. An Infra-red laser at 1064 nm delivered powerful pulses of less than 1 ns duration, sent to the skin surface through a lossless fibre. The light backscattered at a given distance was collected and led onto an Avalanche Photodiode, and the mean TOF was measured on a fast sampling scope. A resolution and a reproducibility of a few picoseconds has been achieved. Experiments were carried out on 100 volunteers of both gender, aged from 18 to 65, on 12 different locations. RESULTS: No matter age and gender, important variations of TOF according to the localization were observed: On the inner forearm, an increase from wrist to elbow, and much higher values on the forehead and neck, whether orientation parallel or perpendicular to Langer lines did not appear significant. Ageing appeared to increase the TOF on forehead and neck, while this effect could not be confirmed on the forearm. Usual skin parameters such as elasticity, roughness and TEWL have been compared to TOF on the same location for each volunteer: TOF and skin roughness were significantly anti-correlated, i.e. the TOF got shorter when the Roughness increased, while a striking correlation was observed between TEWL and TOF. CONCLUSION: These results assert the dependence of TOF on the nature of the skin upper layers (roughness, water diffusion) and on the dermis layer (ageing), and show the potential capabilities offered by TOF to investigate deeply into the skin structure. They have to be confirmed through further experiments, involving measurements at shorter wavelengths, at which the light path into the skin is much smaller, to get a distribution of the TOF inside the tissue.

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. METHODS: To combine and compare the advantages of different microscopic imaging modes, characterization studies were carried out by means of a confocal laser scanning microscope (CLSM), a secondary ion mass spectrometric (SIMS) microscope, and an electron energy loss spectrometric (EELS) microscope. In the case of CLSM, the locations of fluorescent signals inside preparations were determined by factor analysis of biomedical image sequences (FAMIS) and selection of image sequences at emission. RESULTS: By CLSM and FAMIS, we distinguished chelated Eu and Texas Red attached to Fe. By SIMS microscopy, we distinguished Eu and Gd of chlorides and chelates and Fe of a ferumoxide. By EELS microscopy, we distinguished Eu and Gd of chlorides. CONCLUSIONS: Analysis of compounds inside correlative specimens by means of CLSM, SIMS, and EELS microscopes provided complementary results.

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. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their spectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.

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. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains. RESULTS: Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis. CONCLUSION: On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.

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. The time-course of the [Mg2+]i change could be well accounted for by a model of longitudinal diffusion. The mean apparent Mg2+ diffusion coefficient (D(app)) was 188+/-9 microm2 s(-1) (n = 16), approximately four times lower than the value measured in vitro. This reduction likely results from the effects of cytoplasmic viscosity and of Mg2+ binding to low affinity static sites. Another series of measurements was performed on fibers that were either partially or completely free of silicone: under these conditions, the time course of the change in [Mg2+]i was in many cases more complex than predicted by simple diffusion.

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. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.

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). Production of these two species is nucleotide (ATP or 2'-dATP)- and pH-dependent. The chromatographic data presented here could lead to reconsideration of the generally assumed emission mechanism, which involves one emitter only. It could also suggest that each free emitting species is related to a colour of emission corresponding to the two defined wavelengths previously described ( approximately 575 and approximately 620 nm).

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. Photoaffinity labelling of the second site by 8-azido-AMP gives similar results. The amplification effect of CoA and acetyl-CoA is also reconsidered according to this model.

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. With tissues such as tendon and cartilage, better results were obtained after mild trypsin treatment, which allowed better isolation and staining of the collagen fibrils. The main difference observed in the Fourier transforms was the presence in type II collagen fibrils of a strong tenth-order peak (corresponding to the tenth harmonic of the fundamental frequency). In order to discriminate between the two collagens, we measured the ratio (R) of the areas under the ninth- and tenth-order peaks. In trypsin treated tissues, the distributions of these ratios were clearly separated: below 1.0 for type II collagen fibrils and above 1.5 for type I collagen fibrils. This method appears to be suitable for rapid typing of type I and II collagen fibrils and might be useful for determining the exact composition of fibrils in tissues, such as intervertebral discs, that contain these both types of collagen.

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. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be 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. A set of intracellular APIII-Mg2+ calibration spectra was established after permeabilization of the cell membrane with saponin. In comparison with recordings obtained in K+ solutions, the APIII absorption spectra recorded on a myoball exhibited a red shift and an overall change in absorbance, similar to that observed in a protein (bovine serum albumin: BSA) solution. The apparent dissociation constant of APIII for Mg2+ in the myoplasm was found to be 3.16 mM, significantly higher than the 1.86 mM measured in K+ solutions at the same pH (7.35). A stoichiometric ratio of 1:1 was found, however, both in solution and in the myoplasm. In addition, the injected APIII significantly affected the [Mg2+]i of myoballs. The [Mg2+]i was found to be 0.9+/-0.18 mM in 85 myoballs, on the basis of the intracellular calibration spectra obtained at the same myoplasmic APIII concentration ( approximately 2.5 mM), and after correction for the perturbing effect of the dye. It is concluded that an intracellular calibration and recording of whole spectrum are essential for proper interpretations of intracellular dye signals.

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. Using a Tyrode solution containing 5 microM tetrodotoxin (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) onto the endplate induced intracellular free calcium transients the mean maximal amplitude of which was 360 +/- 30 nM from a mean resting value of 72 +/- 7 nM (n = 13). In cells bathed with a K(+)-rich solution (145 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca2+]i from a mean resting value of 53 +/- 7 nM to a maximum of 222 +/- 24 nM (n = 33). 3. In cell-attached membrane patches at the endplate membrane of muscle fibres bathed in a K(+)-rich external solution, using a pipette filled with Tyrode solution, external application of 0.1 mM ACh could induce a transient burst opening of channels carrying an outward current of an average amplitude of 4.6 +/- 0.2 pA at 0 mV (n = 8). 4. These channels were characterized as Ca2(+)-activated K+ channels. At 0 mV, in inside-out patches excised from the endplate membrane area, they displayed a conductance of 60 and 224 pS in the presence of Tyrode and K(+)-rich solution in the pipette, respectively. Half-maximum activation was found for a [Ca2+]i close to 4 microM. The channels showed a typical voltage dependence. In outside-out patches these channels were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTX (5 microM), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the application of ACh. 6. Although the physiological relevance of this ACh-induced K+ channel activation remains unclear, results suggest that, in the presence of a physiological extracellular [Ca2+], Ca2+ entry through the endplate nicotinic receptors can produce a local increase in [Ca2+]i, sufficient to trigger the opening of Ca2+-activated K+ channels in the adjacent surface membrane.

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). The method gives a real-time measurement of the most probable value of sarcomere length in one isolated cell with a temporal resolution of 20 ms. When used on a cell attached between two carbon fibers, the auxotonic force developed by the cell upon electrical stimulation can be simultaneously measured together with the SL in various conditions of stretch. Preliminary results have been presented in abstract form (Gannier et al., vol 24, pp. S47, 1992).

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 7.3 (Bernengo and Cardinaud 1982). This discrepancy, which is reexamined in the present report, suggests that in KCl solution the rod portion of the myosin molecule is bent with an average angle close to 110 degrees, whereas in pyrophosphate at elevated pH it assumes a nearly straight and rigid conformation. Electric birefringence shows that the amount of dimeric and polymeric species in pyrophosphate media at pH's 8.0 and 8.5 is certainly greater than usually thought. In these media, relaxation times can be measured correctly at pH 9.0. A comparative analysis of the influence of protein concentration, field strength, medium composition and concentration, pH and temperature showed that a high relaxation time is associated with the presence of pyrophosphate and that the myosin tail is significantly stiffened in the presence of this anion.

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 7.3) the medium currently used for biochemical assays of myosin in its native state as well as of its subfragments. The comparison of myosin and rod relaxation times (17.2 and 22.8 microseconds, respectively) suggests that the average bend angle in the tail is sharper in intact myosin (90 degrees) whereas rod, when detached from the heads, is a more elongated species with an average bend angle of 120-135 degrees. The LMM relaxation time (6.4 microseconds) is consistent with a rigid linear stick model of length 78 nm. Flexibility in myosin tail is thus confirmed as located in the HMM-LMM hinge. LMM and rod did not exhibit any significant variation of their apparent relaxation times with concentration and the decay curves were best fitted by a single exponential, evidence that the concentration of parallel staggered dimers was negligible in the concentration range studied here (0-7 g/l). This observation lends support to previous results obtained with myosin. Respective HMM, LMM, and rod molecular weights and homogeneity as evaluated by SDS-PAGE analysis were correlated to the Kerr constants of their solutions. Large variations in LMM Kerr constants could be related to the loss of a COOH-terminal peptide on prolonged chymotryptic digestion. Electric birefringence combined with depolarized light scattering is presented as a potential method for net charge distribution studies.

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. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.

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 7.04). Solutions were then heated and the birefringence and light scattering followed during the fibrillogenesis reaction. The monomer presents a translational diffusion coefficient of 0.85 X 10(-7) cm2/s and a rotary diffusion coefficient of 1150 +/- 50 s-1; these values are consistent with a rodlike molecular model of 220 +/- 10 nm length and 4 +/- 1 nm diameter, substantially different from electron microscopic values of 290 and 1.5 nm, respectively. We propose that at pH 7.04 and relatively high ionic strength, the collagen monomer unit must exhibit substantial deviation from a completely rigid and extended rodlike structure. During the entire lag phase in a thermally induced fibrillogenesis reaction, the relaxation times for both translational and rotational motion remain virtually unchanged. The monomer polarity is also unchanged, as shown by reverse pulse birefringence data. No intermediate size soluble aggregates, such as dimers or trimers, have been detected between monomer and very large aggregates or fibrils during the process, although early multistep assembly products (dimers, trimers) could have been seen if present. These data suggest a model for fibrillogenesis emphasizing a monomer-related nucleation event, such as internal stiffening or conformational transition, followed by a rapid continuous growth up to large fibrils.