Circular polarization memory in polydisperse scattering media.

We investigate the survival of circularly polarized light in random scattering media. The surprising persistence of this form of polarization has a known dependence on the size and refractive index of scattering particles, however a general description regarding polydisperse media is lacking. Through analysis of Mie theory, we present a means of calculating the magnitude of circular polarization memory in complex media, with total generality in the distribution of particle sizes and refractive indices. Quantification of this memory effect enables an alternate pathway toward recovering particle size distribution, based on measurements of diffusing circularly polarized light.

In vivo determination of optical properties of normal and tumor tissue with white light reflectance and an empirical light transport model during endoscopy.

Determination of tissue optical properties is fundamental for application of light in either therapeutical or diagnostics procedures. In the present work we implemented a spatially resolved steady-state diffuse reflectance method where only two fibers (one source and one detector) spaced 2.5 mm apart are used for the determination of the optical properties. The method relies on the spectral characteristics of the tissue chromophores (water, dry tissue, and blood) and the assumption of a simple wavelength dependent expression for the determination of the reduced scattering coefficient. Because of the probe dimensions the method is suited for endoscopic measurements. The method was validated against more traditional models, such as the diffusion theory combined with adding doubling for in vitro measurements of bovine muscle. Mean and standard deviation of the absorption coefficient and the reduced scattering coefficient at 630 nm for normal mucosa were 0.87+/-0.22 cm(-1) and 7.8+/-2.3 cm(-1), respectively. Cancerous mucosa had values 1.87+/-1.10 cm(-1) and 8.4+/-2.3 cm(-1), respectively. These values are similar to data presented by other authors. Blood perfusion was the main variable accounting for differences in the absorption coefficient between the studied tissues.

Selective photothermal interaction using an 805-nm diode laser and indocyanine green in gel phantom and chicken breast tissue.

Laser immunotherapy, a novel therapy for breast cancer, utilises selective photothermal interaction to raise the temperature of tumour tissue above the cell damage threshold. Photothermal interaction is achieved with intratumoral injection of a laser-absorbing dye followed by non-invasive laser irradiation. When tumour heating is used in combination with immunoadjuvant to stimulate an immune response, antitumour immunity can be achieved. In this study, the selective photothermal effect was investigated using gel phantom and chicken breast tissue. An 805-nm diode laser and indocyanine green (ICG) were used. An ICG-containing gelatin phantom was constructed to simulate targeted tumour tissue. The target gel was buried inside chicken breast tissue and the tissue-gel construct was irradiated by the laser. Temperatures at different locations in the construct were measured during the laser irradiation. For comparison, the thermal effect of an Nd:YAG laser on the tissue-gel construct was also investigated. Selective heating of target gel containing 0.27% ICG and buried 1 cm below the chicken tissue surface was achieved with the 805-nm diode laser using a power of 0.85 W and beam radius of 1 cm. The target gel experienced a temperature increase of more then 6 degrees C whereas the surrounding chicken breast tissue experienced only a minor temperature increase. The feasibility of this experimental set-up has been shown. It will be used in the future to optimise treatment parameters such as laser power, laser beam radius, and dye concentration.

Iterative reconstruction algorithm for optoacoustic imaging.

Optoacoustic imaging is based on the generation of thermoelastic stress waves by heating an object in an optically heterogeneous medium with a short laser pulse. The stress waves contain information about the distribution of structures with preferential optical absorption. Detection of the waves with an array of broadband ultrasound detectors at the surface of the medium and applying a backprojection algorithm is used to create a map of absorbed energy inside the medium. With conventional reconstruction methods a large number of detector elements and filtering of the signals are necessary to reduce backprojection artifacts. As an alternative this study proposes an iterative procedure. The algorithm is designed to minimize the error between measured signals and signals calculated from the reconstructed image. In experiments using broadband optical ultrasound detectors and in simulations the algorithm was used to obtain three-dimensional images of multiple optoacoustic sources. With signals from a planar array of 3x3 detector elements a significant improvement was observed after about 10 iterations compared to the simple radial backprojection. Compared to conventional methods using filtered backprojection, the iterative method is computationally more intensive but requires less time and instrumentation for signal acquisition.

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity.

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.

Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer.

Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs. In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined. Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+. H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order. Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting. Incubation of HSD with stereospecifically deuterated NADP[2H] and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde. The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.

Substrate-assisted catalysis of the PAR1 thrombin receptor. Enhancement of macromolecular association and cleavage.

Platelet activation and aggregation are mediated by thrombin cleavage of the exodomain of the PAR1 receptor. The specificity of thrombin for PAR1 is enhanced by binding to a hirudin-like region (Hir) located in the receptor exodomain. Here, we examine the mechanism of thrombin-PAR1 recognition and cleavage by steady-state kinetic measurements using soluble PAR1 N-terminal exodomains. We determined that the primary role of the PAR1 Hir sequence is to reduce the kinetic barriers to formation of the docked thrombin-PAR1 complex rather than to form high affinity ground-state interactions. In addition, the exosite I-bound Hir motif facilitates the productive interaction of the PAR1 (38)LDPR/SFL(44) sequence with the active site of thrombin. This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin. This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence.

Modeling photon transport in transabdominal fetal oximetry.

The possibility of optical oximetry of the blood in the fetal brain measured across the maternal abdomen just prior to birth is under investigation. Such measurements could detect fetal distress prior to birth and aid in the clinical decision regarding Cesarean section. This paper uses a perturbation method to model photon transport through an 8-cm-diam fetal brain located at a constant 2.5 cm below a curved maternal abdominal surface with an air/tissue boundary. In the simulation, a near-infrared light source delivers light to the abdomen and a detector is positioned up to 10 cm from the source along the arc of the abdominal surface. The light transport [W/cm2 fluence rate per W incident power] collected at the 10 cm position is Tm = 2.2 x 10(-6) cm(-2) if the fetal brain has the same optical properties as the mother and Tf = 1.0 x 10(-6) cm(-2) for an optically perturbing fetal brain with typical brain optical properties. The perturbation P=(Tf - Tm)/Tm is -53% due to the fetal brain. The model illustrates the challenge and feasibility of transabdominal oximetry of the fetal brain.

A study of aminolevulinic acid-induced protoporphyrin IX fluorescence kinetics in the canine oral cavity.

BACKGROUND AND OBJECTIVE: 5-Aminolevulinic acid-induced protoporphyrin IX is a promising photosensitizer that could enhance the spectroscopic contrast between normal and diseased oral tissues. Knowledge of the pharmacokinetics and effects on tissue type are important for diagnostic and therapeutic procedures. STUDY DESIGN/MATERIALS AND METHODS: Dogs randomly were administered five doses of 5-aminolevulinic acid: 5, 25, 50, 75, and 100 mg/kg. The fluorescence was recorded from buccal mucosa, gums, tongue, and facial skin using a fiberoptic probe connected to an optical multichannel analyzer. Blood samples were collected for hematologic and serum biochemical analysis. Pharmacokinetic parameters of interest were estimated using a compartmental model. RESULTS: Protoporphyrin fluorescence at all sites reached a peak in 2-6 hours, and returned to baseline in 24-31 hours, depending on the dose. Plasma protoporphyrin peaked earlier than oral tissues. CONCLUSION: The rate of synthesis of protoporphyrin, and its conversion to heme products are dose dependent. Different tissues have different pharmacokinetic response.

Source of error in calculation of optical diffuse reflectance from turbid media using diffusion theory.

Diffusion theory and similarity relations were used to calculate the optical diffuse reflectance of an infinitely narrow laser beam incident upon a semi-infinite turbid medium. The results were analyzed by comparison with the accurate results from Monte Carlo simulations. Because a large number of photon packets were traced, the variance of the results from Monte Carlo simulations was small enough to reveal the detailed defects of the diffusion theory and the similarity relations, which are broadly used in photomedicine. We demonstrated that both diffusion theory and similarity relations provide very accurate results when the photon sources are isotropic and buried more deeply than one transport mean free path in turbid media. We found that the key factor affecting the accuracy of the diffusion theory application was the conversion from the infinitely narrow laser beam to an isotropic point source in turbid media.

Imaging superficial tissues with polarized light.

OBJECTIVE: Polarized light can be used to obtain images of superficial tissue layers such as skin, and some example images are presented. This study presents a study of the transition of linearly polarized light into randomly polarized light during light propagation through tissues. STUDY DESIGN/MATERIALS AND METHODS: The transition of polarization was studied in polystyrene microsphere solutions, in chicken muscle (breast) and liver, and in porcine muscle and skin. The transition is discussed in terms of a diffusion process characterized by an angular diffusivity (radians(2)/mean free path) for the change in angular orientation of linearly polarized light per unit optical path traveled by the light. RESULTS: Microsphere diffusivity increased from 0.031 to 0.800 for diameters decreasing from 6.04 microm to 0.306 microm, respectively. Tissue diffusivity varied from a very low value (0.0004) for chicken liver to an intermediate value (0.055) for chicken and porcine muscle to a very high value (0.78) for pig skin. CONCLUSION: The results are consistent with the hypothesis that birefringent tissues randomize linearly polarized light more rapidly than nonbirefringent tissues. The results suggest that polarized light imaging of skin yields images based only on photons backscattered from the superficial epidermal and initial papillary dermis because the birefringent dermal collagen rapidly randomizes polarized light. This anatomic region of the skin is where cancer commonly arises.

Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae.

Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms. Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 A. The resolution limit for these crystals is 3.9 A. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212. The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 A, and the observed resolution limit is 2.2 A. Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 A, beta = 91.9 degrees. This last crystal form has a diffraction limit of 2.7 A resolution.

Light distributions from point, line and plane sources for photochemical reactions and fluorescence in turbid biological tissues.

Light distributions in biological tissues are summarized in simple expressions for spherical, cylindrical and planar geometries due to point sources, line sources and planar sources. The goal is to provide workable tools for computing light distributions that govern the amount and distribution of photochemical reactions in experimental solutions, films and biological tissues. Diffusion theory expressions are compared with Monte Carlo simulations. Analytic expressions that mimic accurate Monte Carlo simulations are presented. Application to fluorescence measurements and prediction of necrotic zones in photodynamic therapy are outlined.

Path integral description of light transport in tissue.

The early photons that arrive at a collector through a large thickness of tissue have potential for imaging internal organ structure, function, and status with improved image resolution relative to late arriving photons which have been diffusely scattered. Imaging algorithms require a theory to calculate early photon arrival for comparison with experimental data. The path integral description of light transport describes the movement of photons as particles undergoing collisions in a scattering medium based on the Brownian motion formalism of Feynman and Hibbs (unconstrained path) which applies the principle of least action. Including the additional constraint that photons have a constant velocity of c yields paths that conserve the speed of light (constrained path). This paper outlines the basic derivation of the path integral method and compares the constrained and unconstrained paths.

Acoustically modulated speckle imaging of biological tissues.

A video laser speckle imaging technique yields images with contrast based on the mechanical properties of a tissue. Fluctuations of laser speckle patterns induced by acoustically driving the tissue at various frequencies in the 0-30-Hz range encode the mechanical strain of the tissue. At each acoustic frequency and within the camera acquisition time, each camera pixel integrates a temporally fluctuating speckle intensity whose variance encodes the mechanical strain in response to the acoustic modulation. The magnitude and the frequency dependence of this strain provide mechanical information about the tissue and are the contrast mechanism for images.

Diagnostic potential of laser-induced autofluorescence emission in brain tissue.

Laser-induced autofluorescence measurement of the brain was performed to assess its spectroscopic properties and to distinguish brain tumors from the normal tissues. The excitation-induced emission spectra were plotted on a 2-dimensional map, the excitation-emission matrix, to determine the excitation wavelengths most sensitive for the spectroscopic identification of brain tumors. The excitation-emission matrices of various types of human brain tumors and normal brain samples lead to the selection of three fluorescence peaks at 470, 520, and 630 nm, corresponding excitation light at 360, 440, and 490 nm, respectively for comparing the autofluorescence signatures of brain tissue. The fluorophores most likely related to each of these peaks are NAD(P)H, various flavins, and porphyrins, respectively. In vivo studies of rat gliomas showed that "NAD(P)H", "flavin", and "porphyrin" fluorescence were lower in gliomas than in normal brain. This finding suggests that there are certain relationship between brain tissue autofluorescence intensity and metabolic activity. In vitro human normal brain tissue fluorescence signals were lower in gray matter than in white matter and "NAD(P)H" fluorescence were lower in all measured human brain tumors than in normal brain. "Flavin" and "porphyrin" fluorescence in the neoplastic tissues was lower or higher than normal tissue depending on their nature. In conclusion, the fluorescence spectroscopic diagnostic system might be able to distinguish brain tumors from the normal brain tissue. The results of this study need to be verified and the investigation extended to human brain tumors in the operating room.

Perturbation theory for diffuse light transport in complex biological tissues.

A perturbation theory for the forward problem of optical transport in turbid media is developed. It is applicable to media with scattering and absorption in homogeneties and steady-state and modulated light. Absorbing perturbations can be described by a volume distribution of virtual sources that primarily causes a monopole perturbation light field. Scattering objects have an additional contribution that, in the limiting case of sharply bounded objects, is represented by a surface distribution of virtual sources and causes a dipolelike perturbation pattern. Using the concept of virtual sources, we discuss a possible ambiguity between the perturbations from scattering and absorbing inhomogeneities and the implications for the source-detector placement in inverse problems. We show that the surface effects due to sharp boundaries of scattering objects pose both a numerical problem and a chance to improve the resolution of inverse algorithms.

Measurement of tissue optical properties by the use of oblique-incidence optical fiber reflectometry.

Fiber-optic-based oblique-incidence reflectometry is a simple and accurate method for measuring the absorption and reduced scattering coefficients mu(a) and mu?(s) of semi-infinite turbid media. Obliquely incident light produces a spatial distribution of diffuse reflectance that is not centered about the point of light entry. The amount of shift in the center of diffuse reflectance is directly related to the medium's diffusion length D. We developed a fiber-optic probe to deliver light obliquely and sample the relative profile of diffuse reflectance. Measurement in absolute units is not necessary. From the profile, it was possible to measure D, perform a curve fit for the effective attenuation coefficient mu(eff), and then calculate mu(a) and mu?(s). This method was verified with Monte Carlo simulations and tested on tissue phantoms. Our measurements of D and mu(eff) had an accuracy of approximately 5%, thus giving us 10% and 5% accuracy for mu(a) and mu?(s), respectively.

Measurement of tissue optical properties by time-resolved detection of laser-induced transient stress.

We report on a technique utilizing time-resolved detection of laser-induced stress transients for the measurement of optical properties in turbid media specifically suitable for biological tissues. The method was tested initially in nonscattering absorbing media so that it could be compared with spectrophotometry. The basis of this method is provided by the conditions of temporal stress confinement in the irradiated volume where the pressure generated in tissues heated instantly by laser pulses is proportional to the absorbed laser energy density, and the exponential profile of the initial stress distribution in the irradiated volume corresponds to the z-axial distribution of the absorbed laser fluence. Planar thermoelastic waves can propagate in water-containing media with minimal distortion, and their axial profiles can be detected by an acoustic transducer with sufficient temporal resolution. The acoustic waves induced by 14-ns laser pulses in nonscattering media, turbid gels, and tissues were measured by a piezoelectric transducer with a 3-ns response time. Temporal profiles of stress transients yielded z-axial distributions of the absorbed laser energy in turbid and opaque media, provided that the speed of sound in these media was known. The absorption and effective scattering coefficients of beef liver, dog prostate, and human aortic atheroma at three wavelengths, 1064 nm (in near infrared), 532 nm (visible), and 355 nm (near UV), were deduced from laser-induced stress profiles with additional measurements of total diffuse reflectance.

CONV--convolution for responses to a finite diameter photon beam incident on multi-layered tissues.

A convolution program (CONV) solving responses to a collimated finite diameter photon beam perpendicularly incident on a multi-layered tissue has been coded in ANSI Standard C, hence, the program can be executed on various computers. The program, employing an extended trapezoidal rule for integration, convolves the responses to an infinitely narrow photon beam computed by a companion program (MCML). Dynamic data allocation is used for CONV as well as MCML, therefore, the number of tissue layers and grid elements of the grid system can be varied at run time. The potential error due to not scoring the first photon-tissue interactions separately is illustrated. The program, including the source code, has been in the public domain since 1992 and can be downloaded from the web site at http:(/)/biomed.tamu.edu/-lw.