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At least 973 records · Page 54Linked to original sources
PubMed2001

Refractive-index-mismatch induced aberrations in single-photon and two-photon microscopy and the use of aberration correction.

We examine the effects of aberrations induced by a refractive index mismatch on the signal level and resolution of single-photon (1-p) and two-photon (2-p), conventional and confocal scanning microscopes. In particular, we consider the aberrations introduced by an interface between oil/glass and water. Resolution is defined in terms of enclosed fluorescence, rather than full-width half-maximum, revealing more useful information for heavily aberrated point spread functions (PSFs). It is shown that, at large focusing depths, the resolution of 2-p conventional and 1-p confocal microscopes are almost identical. The benefits of aberration correction are examined by removing Zernike aberration modes. With aberration correction, the best resolution is found for 1-p confocal and 2-p confocal modes. An approximation based upon geometrical optics is also introduced which shows that the axial resolution of heavily aberrated PSFs is roughly proportional to focusing depth.

Microscopy, Fluorescence↗
PubMed2001

Optical characterization of melanin.

The optical properties of melanin have been characterized for a number of laser wavelengths in the visible region. The index of refraction of melanin is measured by the conventional method of minimum deviation using a hollow quartz prism at these wavelengths. The inverse adding doubling method based on the diffusion approximation and radiative transport theory have been employed to determine the absorption, scattering, and scattering anisotropy coefficients of melanin from the measurements of diffuse transmission, diffuse reflection and collimated transmission using double integrating spheres. The results obtained by the use of inverse adding doubling method have been compared to the Monte Carlo simulation technique.

Animals↗
PubMed2001

Experimental verification of the effect of refractive index mismatch on the light fluence in a turbid medium.

Diffusion theory is often used to model the transport of light within tissue. It can be used to calculate the light fluence rate in tissue, for example, during photodynamic therapy, or to measure the absorption and scattering properties of tissue. For both of these applications, the influence of the interface between the tissue and the exterior medium on the fluence rate inside the tissue must be known in order to make accurate calculations. We present an experimental investigation of the effect of the refractive index mismatch at the tissue interface on the internal light fluence rate and on the spatially resolved diffuse reflectance as the boundary conditions of the tissue/external medium are changed. The effects of changing the relative refractive index at the boundary are compared to predictions of diffusion theory. The effect of the refractive index mismatch is predicted correctly by diffusion theory.

Animals↗
PubMed2002

Simultaneous refractive index and thickness measurements of bio tissue by optical coherence tomography.

This paper presents two techniques based on optical coherence tomography, the "focus tracking method" and the "optical path shifting method," for determining refractive index and thickness simultaneously, which are especially useful for bio tissues. From comparison of these two methods, it was found that the focus tracking method is suitable for in vivo measurement, but does not have high precision. The optical path shifting method is limited to in vitro measurement, but has high precision. Using the optical path shifting method, the refractive indices of cucumber were measured at the wavelength of 850 and 1300 nm.

Fiber Optic Technology↗
PubMedDate not supplied

New optical scheme for a polarimetric-based glucose sensor.

A new optical scheme to detect glucose concentration in the aqueous humor of the eye is presented. The ultimate aim is to apply this technique in designing a new instrument for, routinely and frequently, noninvasively monitoring blood glucose levels in diabetic patients without contact (no index matching) between the eye and the instrument. The optical scheme exploits the Brewster reflection of circularly polarized light off of the lens of the eye. Theoretically, this reflected linearly polarized light on its way to the detector is expected to rotate its state of polarization, owing to the presence of glucose molecules in the aqueous humor of a patient's eye. An experimental laboratory setup based on this scheme was designed and tested by measuring a range of known concentrations of glucose solutions dissolved in water. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

Aqueous Humor↗
PubMedDate not supplied

In vivo birefringence and thickness measurements of the human retinal nerve fiber layer using polarization-sensitive optical coherence tomography.

Glaucoma causes damage of the nerve fiber layer, which may cause loss of retinal birefringence. Therefore, PS-OCT is a potentially useful technique for the early detection of glaucoma. We built a fiber-based PS-OCT setup that produces real-time images of the human retina in vivo, coregistered with retinal video images of the location of PS-OCT scans. Preliminary measurements of a healthy volunteer show that the double-pass phase retardation per unit of depth of the RNFL is not constant and varies with location, with values between 0.18 and 0.37 deg/microm. A trend in the preliminary measurements shows that the nerve fiber layer located inferior and superior to the optic nerve head is more birefringent than the thinner layer of nerve fiber tissue in the temporal and nasal regions.

Adult↗
PubMedDate not supplied

Digital image capture and quantification of subtle lens opacities in rodents.

A rapid, sensitive, and cost-effective method is reported for the subjective and objective documentation of subtle opacities in lenses of unanesthetized transgenic mice or selenite-injected rat pups as models for cataract formation. Animal eyes were dilated with eye drops and the animal was positioned in front of a Nikon FS2 photo slit lamp. Slit-lamp observations were recorded using a Canon Optura Pi digital video recorder. High-quality images of opacifying lenses were captured from the video and quantified using densitometry at progressive stages of opacification. In mice, targeted genomic deletion of the proteins CP49 (a lens-specific filament) or Six5 (a model for myotonic dystrophy) resulted in subtle cataracts that were easily recorded and quantified using this instrumentation. In rats, the early progressive changes leading to a dense nuclear opacity caused by selenite injection were easily documented using this instrumentation. Low-cost components combined with a conventional slit-lamp ophthalmoscope were used to capture high-quality images of selected stages of cataract formation for quantitative analysis using commercial software.

Animals↗
PubMedDate not supplied

Amending of fluorescence sensor signal localization in human skin by matching of the refractive index.

Fluorescence diagnostic techniques are notable amongst many other optical methods because they offer high sensitivity and noninvasive measurement of tissue properties. However, a combination of multiple scattering and physical heterogeneity of biological tissues hampers interpretation of the fluorescence measurements. Analyses of the spatial distribution of endogenous and exogenous fluorophores excitation within tissues and their contribution to the detected signal localization are essential for many applications. We have developed a novel Monte Carlo technique that gives a graphical perception of how the excitation and fluorescence detected signal are localized in tissues. Our model takes into account the spatial distribution of fluorophores, the variation of concentrations and quantum yield. We demonstrate that matching the refractive indices of the ambient medium and topical skin layer improves spatial localization of the detected fluorescence signal within the tissues.

Computer Simulation↗
PubMedDate not supplied

New method for evaluation of in vivo scattering and refractive index properties obtained with optical coherence tomography.

Optical coherence tomography (OCT) provides more parameters than pure morphology does. In a recent paper [A. Knuettel and M. Boehlau-Godau, J. Biomed. Opt. 5(1) 83-92 (2000)] we have shown that the refractive index (RI) can be evaluated in a localized manner in skin tissue under in vivo conditions. Based on a theory, originally developed for light detecting and ranging applications [L. Thrane et al., J. Opt. Soc. Am. A 17(3) 484-490 (2000)], the parameter mean scattering angle (MSA) could be derived in addition to RI. The effects of hydration on MSA and RI have been evaluated in vitro in pigskin and in vivo in human skin with our OCT scanner SkinDex 300. These parameters may have a viable impact in (cosmetic) skin research and clinical diagnoses. To the best of our knowledge, this is the first time that (multiple) scattering of light has been quantified through the observation of a new scattering parameter under in vivo conditions.

Adult↗
PubMedDate not supplied

Optical characterization of bovine retinal tissues.

An in-depth characterization of the optical properties of bovine retinal and retinal pigment epithelium-choroidal tissues has been performed. The indices of refraction of these ocular tissues were determined by applying Brewster's law. The inverse adding doubling method based on the diffusion approximation and radiative transport theory is applied to the measured values of the total diffuse transmission, total diffuse reflection, and collimated transmission to calculate the optical absorption, scattering, and scattering anisotropy coefficients of the bovine retinal and retinal pigment epithelium-choroidal tissues. The values of the optical properties obtained from the inverse adding doubling method are compared with those generated by the Monte Carlo simulation technique. Optical polarization measurements are also performed on bovine retinal tissues. Our studies show that both retina and retinal pigment epithelium-choroid possess strong polarization characteristics.

Animals↗
PubMedDate not supplied

Constrained optimization of Drude's equations eliminates effects of confounding molecules for the polarimetric measurement of glucose.

Common confounding factors for polarimetric concentration measurements include additional optical rotations from unknown optically active molecules, linear birefringence of the medium, and path length variability. We show that by approximating Drude's equation and taking several measurements from the same sample at different wavelengths, the error due to confounding rotations in the measurements can theoretically be canceled. The analysis is developed with regard to glucose sensing in aqueous humor. First, we show that the optical rotatory dispersions of the known molecules in bovine aqueous humor could be represented by Drude's equations. Then, the total optical rotation is approximated by a function combining Drude's equations for the major contributors in the sample, i.e., glucose, glutamine, fructose, and phenylalanine. The concentration-related unknown coefficients in the approximating function are found by constrained nonlinear optimization of the function at different wavelengths. This technique is tested on a published data set and four alterations of those data: (1) concentrations randomly varied within narrow limits, (2) similar to alteration 1 but with significantly elevated glucose concentration, (3) similar to alteration 1 but with significantly decreased glucose concentration, and (4) concentrations randomly varied within wider limits than alteration 1. The method produces very accurate glucose-concentration estimates in all of these data sets. The relative error was smaller than 1% in all except the low-glucose sample (1.4%). This method may prove useful in noninvasive glucose measurement in humans.

Algorithms↗
PubMedDate not supplied

Surface plasmon resonance phase-shift interferometry: real-time DNA microarray hybridization analysis.

Surface plasmon resonance (SPR) phase-shift interferometry (PSI) is a novel technique which combines SPR and modified Mach-Zehnder PSI to measure the spatial phase variation caused by biomolecular interactions upon a sensing chip. The SPR-PSI imaging system offers high resolution and high-throughout screening capabilities for microarray DNA hybridization without the need for additional labeling, and provides valuable quantitative information. The SPR-PSI imaging system has an enhanced detection limit of 2.5 x 10(-7) refraction index change, a long-term phase stability of pi/100 in 30 min, and a spatial phase resolution of pi/300 with 100 x 100 microm2 detection area. This study successfully demonstrates the label-free observation of 15-mer DNA microarray.

DNA↗
PubMedDate not supplied

Scattering coefficient determination in turbid media with backscattered polarized light.

A simple empirical method is presented to determine the scattering coefficient mu' s from backscattered polarized images of turbid media. It uses the ratio, pixel by pixel, of two images that are the second and the first backscattered Stokes parameter images Q and I, respectively. Taking this image ratio, then integrating it over the azimuth angle, we get a function depending on the distance from the light entrance point. This function has a maximum. Using Monte Carlo simulations, for a fixed reduced scattering coefficient mu s and for an anisotropy factor g varying between 0 and 0.8, it is found a linear relationship between the scattering coefficient mu s and the inverse of the maximum position of this function.

Algorithms↗
PubMedDate not supplied

Light transport in two-layer tissues.

We study theoretically light backscattered by tissues using the radiative transport equation. In particular we consider a two-layered medium in which a finite slab is situated on top of a half space. We solve the one-dimensional problem in which a plane wave is incident normally on the top layer and is the only source of light. The solution to this problem is obtained formally by imposing continuity between the solutions for the upper and lower layers. However, we are interested solely in probing the top layer. Assuming that the optical properties in the lower layer are known, we remove it from the problem yielding a finite slab problem by prescribing an alternate boundary condition. This boundary condition is derived using the theory of Green's functions and is exact. Hence, one needs only to solve the transport equation in a finite slab using this alternate boundary condition. We derive an asymptotic solution for the case when the slab is optically thin. We extend these results to the three-dimensional problem using Fourier transforms. These results are validated by comparisons with numerical solutions for the entire two-layered problem.

Biological Transport↗
PubMedDate not supplied

Oblique-incidence illumination and collection for depth-selective fluorescence spectroscopy.

Optimization of device-tissue interface parameters may lead to an improvement in the efficacy of fluorescence spectroscopy for minimally invasive disease detection. Although illumination-collection geometry has been shown to have a strong influence on the spatial origin of detected fluorescence, devices that deliver and/or collect light at oblique incidence are not well understood. Simulations are performed using a Monte Carlo model of light propagation in homogeneous tissue to characterize general trends in the intensity and spatial origin of fluorescence detected by angled geometries. Specifically, the influence of illumination angle, collection angle, and illumination-collection spot separation distance are investigated for low and high attenuation tissue cases. Results indicate that oblique-incidence geometries have the potential to enhance the selective interrogation of superficial or subsurface fluorophores at user-selectable depths up to about 0.5 mm. Detected fluorescence intensity is shown to increase significantly with illumination and collection angle. Improved selectivity and signal intensity over normal-incidence geometries result from the overlap of illumination and collection cones within the tissue. Cases involving highly attenuating tissue produce a moderate reduction in the depth of signal origin. While Monte Carlo modeling indicates that oblique-incidence designs can facilitate depth-selective fluorescence spectroscopy, optimization of device performance will require application-specific consideration of optical and biological parameters.

Algorithms↗
PubMedDate not supplied

Refractive index of tissue measured with confocal microscopy.

Refractive index of tissue is an essential parameter in many bio-optical experiments, yet little data can be found in literature. Several methods have been proposed to measure refractive index in tissue samples, but all have specific limitations, such as low accuracy, the need for large amounts of tissue, or the complexity of the measurement setup. We propose a new method using a standard confocal microscope and requiring only small tissue samples. A thin slice of tissue is put next to a layer of immersion fluid of exactly the same thickness. The actual thickness of the fluid layer is directly measured with the microscope, as there is no refractive index mismatch. A difference between index of refraction of the tissue and of the immersion medium causes an axial scaling factor. The optical thickness of the specimen is thus measured with the microscope, and as its actual thickness equals the known thickness of the fluid layer, the axial scaling factor is readily determined. From this factor, we calculate the refractive index of the tissue. We use a diffraction model to take the point spread function (PSF) of the microscope into account, so we can determine the index of refraction to a very high accuracy. We demonstrate the method on bovine muscle tissue and find a value of n=1.382+/-0.004, at 592 nm.

Algorithms↗
PubMedDate not supplied

Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging.

Often fluorophores observed by microscopy are located close to a planar surface as in total internal reflection microscopy or single molecule studies. The optical properties of fluorescence excitation and emission near a planar surface (possibly metal film coated) between two dielectrics are well understood from the viewpoint of classical electromagnetic theory. We present an experimental method whereby the angular dependence of the emission pattern of a fluorophore near a bare surface or a metal film coated surface that supports surface plasmon resonance can be measured with microscope optics. The technique involves an alteration of the microscope optics to directly record (on a digital CCD camera) the intensity pattern at the objective's back focal plane, which directly maps the angular emission pattern of fluorescence. The experimental emission profile on both glass and aluminum-coated surfaces is anisotropic with a peak at either the critical angle or both the critical angle and the surface plasmon angle. The observed profiles agree well with computer calculations and suggest some optical modifications that are potentially useful in cell biophysics.

Coated Materials, Biocompatible↗
PubMedDate not supplied

Polarimetric imaging of subsurface tissue-numerical results.

Tissue images obtained at deeper depths lack significant contrast. To enhance the contrast of these images and to increase the visibility of subsurface tissues, a method is proposed. This technique is based on the principle that photons at longer wavelengths penetrate deeper than photons at shorter wavelengths. In this technique, images in the original and orthogonal polarized states are recorded with the source illumination in linear polarized state at two different wavelengths. Image subtraction of a fraction of the copolarized image from the perpendicular polarized state is done on these images. The images obtained after the first processing are subjected to the next image subtraction where the shorter wavelength image is subtracted from the longer wavelength image. Monte Carlo simulations show that the resultant image has marked contrast up to 2.5 cm.

Algorithms↗