Fluorescence correlation spectroscopy is used extensively for quantitative characterization of biomolecules at very low concentration. However, light aberration and scattering from the tissues are two major factors that affect the results strongly. Although adaptive optics arrangement can correct the aberrations of light to some extent, it fails completely to eliminate the light scattering effect. Recently, exploiting the fact that autocorrelation of a speckle pattern is a sharply peaked point spread function and the optical memory effect, non-invasive imaging of fluorescent sample through a scattering medium has been possible. However, it is also very challenging to measure the dynamic properties of the fluorescent molecules or particles through a scattering layer due to poor signal to noise ratio. In this study, we employ a modality based on speckle cross-correlation enabled via optical memory effect to study two dimensional (2D) diffusion of fluorescent particles hidden behind a scattering film. We realized a 2D diffusing model system by confining fluorescent polystyrene beads of 1µm diameter at the water/air interface behind a TiO2 diffuser. The experimental set up was built up in an epifluorescence configuration. The fluorescent beads were excited by an illumination speckle generated by the incident light in a plane-wave geometry while passing through the disordered TiO2 film. Similarly, the emitted fluorescent signal also traversed through the same TiO2 film to generate the detection speckle, which was eventually recorded by a high frame rate CMOS camera. The experimental set up has also been modelled numerically, where speckle pattern has been generated by a spherical wave, transmitted through a scattering object in an optical microscope. Moreover, the dependence of the speckle size on the numerical aperture, magnification, and the distance of the focal plane from the bead plane has also been studied. The numerical results have been compared with the experimental values to estimate the speckle size. Furthermore, we have evaluated the 2D diffusion constant by monitoring the widening of the 2D speckle cross-correlation function versus lag time. This result has been compared with that obtained with the single particle tracking method without the scattering layer. Quantitative agreement between the results obtained by the speckle cross-correlations and the single particle tracking technique without the diffuser establishes the potential application of this technique in correlation spectroscopy. Superimposed multiple beads speckle patterns were also studied and the results will be presented in the conference.
To evaluated capabilities of multispectral TD-DOT systems in reflection geometry, we
performed a measurement campaign on multimaterial composition phantoms. Results show correct
composition gradation of inclusions but still lack absolute accuracy.
Near-infrared diffuse optical tomography (DOT) is a medical imaging which gives the distribution of the optical properties of biological tissues. To obtain endogenous chromophore features in the depth of a scattering medium, a multiwavelength/time-resolved (MW/TR) DOT setup was used. Reconstructions of the three-dimensional maps of chromophore concentrations of probed media were obtained by using a data processing technique which manages Mellin-Laplace Transforms of their MW/TR optical signals and those of a known reference medium. The point was to
put a constraint on the medium absorption coefficient by using a material basis composed of a given set of chromophores of known absorption spectra. Experimental measurements were conducted by injecting the light of a picosecond near-
infrared laser in the medium of interest and by collecting, for several wavelengths and multiple positions, the backscattered light via two fibers (with a source-detector separation of 15 mm) connected to fast-gated single-photon
avalanche diodes (SPAD) and coupled to a time-correlated single-photon counting (TCSPC) system. Validations of the method were performed in simulation in the same configuration as the experiments for different combination of chromophores. Evaluation of the technique in real conditions was investigated on liquid phantoms composed of an
homogenous background and a 10 mm depth inclusion formed of combination of intralipid and inks scanned at 30
positions and at three wavelengths. Both numerical and preliminary phantom experiments confirm the potential of this method to determine chromophore concentrations in the depth of biological tissues.
Kawata and Tani's [4] experiments showed that the evanescent field created on the surface of an ion exchanged
waveguide could trap and move microparticles. This opened up the possibility of combining conventional optical
trapping with integrated optics in order to create new microsystems for the manipulation of particles or biological
objects. Recently, the use of strip silicon nitride waveguides increased the performances of these systems enabling
higher particles speeds and reduced guided power [12].
Our experiments demonstrate that polarization affects drastically the way particles are propelled along the waveguide
surface. For example in TM polarization, 0,6 &mgr;m diameter gold particles are moving along the center of the waveguide
whereas in TE, they are propelled along its sides. Moreover, it appears that gradient forces involved in this phenomenon
depend on the particle size.
To understand this behavior, a numerical approach of the problem based on the finite element method has been
developed. This method enables the calculation of the 3D distribution of the electric fields. The resulting optical forces
are calculated thanks to the Maxwell stress tensor formalism.
This first experimental and theoretical illustration of repulsive gradient forces on metallic particles opens up perspectives
for polarization based sorting systems.
Fluorescence Correlation Spectroscopy (FCS) is an attractive method to measure molecular concentration, mobility parameters and chemical kinetics. However its ability to descriminate different diffusing species needs to be improved. Recently, we have proposed a simplified spatial Fluorescence cross Correlation Spectroscopy (sFCCS) method, allowing, with only one focused laser beam to obtain two confocal volumes spatially shifted. Now, we present a new sFCCS optical geometry where the two pinholes, a ring and core, are encapsulated one in the other. In this approach all physical and chemical processes that occur in a single volume, like singlet-triplet dynamics and photobleaching, can be eliminated; moreover, this new optical geometry optimises the collection of fluorescence. The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are presented, in addition to the effect of the size of fluorescent particules (nano-beads, diameters : 20, 100 and 200 nm). The relative simplicity of the method leads us to propose sFCCS as an appropriate method for the determination of diffusion parameters of fluorophores in solution or cells. Nevertheless, progresses in the ingeniering of the optical Molecular Detection Efficiency volumes are highly desirable, in order to improve the descrimination between the cross correlated volumes.
Fluorescence fluctuation spectroscopy is applied to study molecules, passing through a small observation volume, usually subjected to diffusive or convective motion in liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or imbedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres), DNA-chips, and dye-stained histidine molecules anchored on silanized glass surfaces.
KEYWORDS: Brain, Magnetism, Near infrared spectroscopy, Heart, Electrocardiography, Absorption, Data acquisition, Linear filtering, Blood circulation, Indocyanine green
Near infrared spectroscopy using either broad band reflectance spectrophotometry or monochromatic illumination has been carried out to monitor non invasively the changes of the concentrations of chromophores in rat brain induced by the intravenous injection of various contrast agents (indocyanine green, ultrasmall magnetic particles suspension, albumine, dextran, or saline solution alone). Depending of the wavelength and of the absorption spectrum of the injected compound the bolus can be seen either by a decrease or an increase of the transmitted light, this latter due to the induced dilution of the blood by the bolus. We suggest that this could be used to determine the arterial input function of the contrast agent needed to perform absolute cerebral blood flow imaging by nuclear magnetic resonance.
Broad band light absorption spectroscopy in the visible range (520-590nm) has been carried out using implanted small optical fibers to probe the hemodynamics of deep tissues (striatum) in rat brain subjected to hypoxia. We observe a decrease of the cerebral blood oxygenation by a factor of up to two, while the cerebral blood volume (CBV) does not seem to increase significantly. However, nuclear magnetic resonance measurements of CBV in the same conditions using a magnetic contrast agent show that CBV increases by about 50%. This shows that absorption spectroscopy in the visible range strongly underestimates the CBV, probably due to the confinement of blood in vessels. This effect is confirmed by absorption spectroscopy measurements performed in phantoms with similar geometry.
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