Localization and characterization of tissue changes by laser backscattering imaging and Monte Carlo simulation

Laser backscattering from biological tissues depends on their composition and blood flow. The onset of the tissue abnormalities is associated with the change in composition at a specific location which may affect laser backscattering. The objective of the present work is to detect the compositional changes in tissue-equivalent phantom of fat, prepared by mixing paraffin wax with wax colors, and to characterize these in terms of their optical parameters. For this purpose these phantoms are scanned by a multi-probe non-contact automatic laser scanning system and images of the normalized backscattered intensity (NBI) are constructed. By scanning the background subtracted image of the phantom the location of the abnormality and its size from the full width at half maximum (FWHM) are determined. The data obtained by ultrasonic technique for localization of the abnormalities are in agreement with that as obtained by the present method. The optical parameters of the abnormality are obtained by matching the measured surface profiles of the abnormality with that of the profile obtained by Monte Carlo simulation. This analysis shows the possibility of detection of changes at the onset stage in tissues as required for planning of the photodynamic therapy.     

Investigation of changes in optical attenuation of bone and neuronal cells in organ culture or three-dimensional constructs in vitro with optical coherence tomography: relevance to cytochrome oxidase monitoring

Changes in optical attenuation, relevant to cytochrome oxidase, of the rat bone periosteal tissue in explanted culture and human neuronal cells in three-dimensional agarose constructs have been monitored by the use of optical coherence tomography (OCT), with potential applications in tissue engineering and diagnosis. A superluminescent diode (SLD) with a peak emission wavelength (lambda = 820 nm) that is the near-infrared absorption band of the oxidized form of CytOx was employed. The attenuation coefficient was obtained from the depth-resolved reflectance profiles of liquid phantoms (naphthol green B with intralipid), explant culture (periosteum of calvaria from rats) and cells in 3D agarose constructs. The absorption coefficient of naphthol green B can be accurately quantified by the linear relationship between attenuation coefficients and the concentration. The difference in the attenuation coefficient of astrocytoma cells in agarose before and after reduction of CytOx is 0.26 +/- 0.10 mm(-1) ( n = 9), whereas no attenuation is observed with the agarose control. Reduction of the enzyme in periosteal tissue leads to a change in attenuation coefficient of 0.43 +/- 0.24 mm(-1) ( n = 7). For comparison, using a biochemical assay, the absorption coefficient of the oxidized-reduced form of CytOx is measured at approximately 8.3 +/- 1.5x10(-3) mm-1 ( n = 4) and 8.7 +/-2.5x10(-3) mm-1 ( n = 4) at 820 nm for astrocytoma cells and rat periosteum, respectively. The lower value of CytOx concentration using biochemical versus OCT measurements may result from shifts in the scattering profile and the amplifying influences of multiple heme-based oxidases, indicating that conventional OCT is not specific enough to monitor redox changes in cytochrome oxidase. However, qualitative shifts in oxidation state are apparent using the technique. Our results suggest the potential application of OCT in providing high-resolution tomographic imaging of tissues in organ culture and cells grown in three-dimensional constructs in vitro.     

Multiprobe laser reflectometry in imaging and characterization of biological tissues

Laser backscattered radiation from human forearm and foot were measured by multi-probe reflectometer, which consisted of one input probe and three output probes placed at distances of 2, 4 and 6 mm from the input probe. The normalized backscattered intensity (NBI) signals from the tissue surface, measured by the output probes, after digitization, were used to reconstruct the reflectance images of tissues in various layers below the skin surface. From NBI profiles measured at various locations of the tissues on the forearm the corresponding optical parameters, the scattering (mu(s)) and absorption (mu(a)) coefficients and the anisotropy parameter g, by matching these with profiles as simulated by Monte Carlo procedure were determined. From these data the optical parametric images of forearm were reconstructed which show the variation of these parameters at various locations. Similarly, the NBI data were collected from the foot sole region of healthy and diabetes subjects and their images reconstructed. These images showed the variation in the NBI in the diabetic foot sole compared to that of healthy subject, indicating the tissue structural changes. These procedures could be useful for diagnostic and therapeutic applications of lasers.     

Optical parameters of embedded abnormalities in tissues as determined by Monte Carlo simulation

Photon propagation through tissue phantoms, made of heart, adipose, and spleen tissues was simulated by Monte Carlo procedure. To detect the presence of deep-seated abnormalities, phantoms of heart with adipose and spleen tissues embedded into it were created and simulations were performed to scan the tissue surface with a source and four detector model. Profiles drawn showed variation in parameters such as backscattered intensity in regions where adipose and spleen tissues were embedded. This study shows that depending on the type of embedded tissue the backscattered fraction as measured at 2 mm from the input fiber is altered. This is enhanced for adipose and decreased for spleen tissue. This is not only shown in scanned profiles on the surface but also in constructed images.     

Optical parameters of embedded abnormalities in tissues as determined by Monte Carlo simulation

Photon propagation through tissue phantoms, made of heart, adipose, and spleen tissues was simulated by Monte Carlo procedure. To detect the presence of deep-seated abnormalities, phantoms of heart with adipose and spleen tissues embedded into it were created and simulations were performed to scan the tissue surface with a source and four detector model. Profiles drawn showed variation in parameters such as backscattered intensity in regions where adipose and spleen tissues were embedded. This study shows that depending on the type of embedded tissue the backscattered fraction as measured at 2?mm from the input fiber is altered. This is enhanced for adipose and decreased for spleen tissue. This is not only shown in scanned profiles on the surface but also in constructed images.     

The influence of the blood vessel diameter on the full scattering profile from cylindrical tissues: experimental evidence for the shielding effect

Optical methods for detecting physiological state based on light–tissue interaction are noninvasive, inexpensive, simplistic, and thus very useful. The blood vessels in human tissue are the main cause of light absorbing and scattering. Therefore, the effect of blood vessels on light–tissue interactions is essential for optically detecting physiological tissue state, such as oxygen saturation, blood perfusion and blood pressure. We have previously suggested a new theoretical and experimental method for measuring the full scattering profile, which is the angular distribution of light intensity, of cylindrical tissues. In this work we will present experimental measurements of the full scattering profile of heterogenic cylindrical phantoms that include blood vessels. We show, for the first time that the vessel diameter influences the full scattering profile, and found higher reflection intensity for larger vessel diameters accordance to the shielding effect. For an increase of 60% in the vessel diameter the light intensity in the full scattering profile above 90° is between 9% to 40% higher, depending on the angle. By these results we claim that during respiration, when the blood‐vessel diameter changes, it is essential to consider the blood‐vessel diameter distribution in order to determine the optical path in tissues.

A CT scan of the measured silicon‐based phantoms. The phantoms contain the same blood volume in different blood‐vessel diameters.     

Chemical weed management in wheat at higher altitudes

To evaluate the effect of different herbicides for controlling weeds in wheat (variety Fakhr-i-Sarhad),at higher attitude, an experiment was conducted at Agriculture Research Station, Chitral during Rabi season 2003-04, using Randomized Complete Block Design, keeping four replications. The experiment, sown in November comprised of eight treatments, viz; seven herbicides and a weedy check. Each treatment consisted of 5 rows each 30 cm apart and 5 m long thus giving a total size of 5 m x 1.5 m. The herbicides used included; terbutryn + triasulfuron at 0.16 kg, 2,4-D at 0.7 kg, fenoxaprop-P-ethyl at 0.93 kg, clodinafop at 0.05 kg, bromoxynil + MCPA at 0.49 kg, carfentrazon-ethyl at 0.02 kg and isoproturon at 1.0 kg a.i ha(-1). The data were recorded on weed kill efficiency (%), fresh weed biomass (kg ha(-1)), plant height (cm), spike length (cm), number of tillers m(-2), number of grains spike(-1), thousand grains weight (g), biological yield (kg ha(-1)), grain yield (kg ha(-1)) and harvest index (%). The data recorded on weed kill efficiency, weed biomass (kg ha1), grains yield (kg ha(-1)) and harvest index (%) were significantly affected by the different herbicidal treatments. Statistically isoproturon treatment exhibited the best performance, with maximum weed kill efficiency (48.26%) and minimum fresh weed biomass (433.3 kg ha(-1)) as compared to weedy check (6 %) and (1102 kg ha(-1)), respectively. Similarly, the spike length (8.34 cm), number of tillers (427 m(-2)), number of grains spike(-1) (38.0), thousand grains weight (39.85 g), biological yield (8475 kg ha(-1)), grain yield (2530 kg ha(-1)) and harvest index (31.3%) were the highest in isoproturon treatments as compared to weedy check having (7.64 cm), (356 m(-2)), (34.1), (37.12 g), (6858 kg), (1913 kg ha(-1)) and (27%), respectively.     

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Composition differences between epicuticular and intracuticular wax substructures: how do plants seal their epidermal surfaces?

The protective wax coating on plant surfaces has long been considered to be non-uniform in composition at a subcellular scale. In recent years, direct evidence has started to accumulate showing quantitative compositional differences between the epicuticular wax (i.e. wax exterior to cutin that can be mechanically peeled off) and intracuticular wax (i.e. wax residing within the mechanically resistant layer of cutin) layers in particular. This review provides a first synthesis of the results acquired for all the species investigated to date in order to assign chemical information directly to cuticle substructures, together with an overview of the methods used and a discussion of possible mechanisms and biological functions. The development of methods to probe the wax for z-direction heterogeneity began with differential solvent extractions. Further research employing mechanical wax removal by adhesives permitted the separation and analysis of the epicuticular and intracuticular wax. In wild-type plants, the intracuticular (1-30 μg cm(-2)) plus the epicuticular wax (5-30 μg cm(-2)) combined to a total of 8-40 μg cm(-2). Cyclic wax constituents, such as triterpenoids and alkylresorcinols, preferentially or entirely accumulate within the intracuticular layer. Within the very-long-chain aliphatic wax components, primary alcohols tend to accumulate to higher percentages in the intracuticular wax layer, while free fatty acids and alkanes in many cases accumulate in the epicuticular layer. Compounds with different chain lengths are typically distributed evenly between the layers. The mechanism causing the fractionation remains to be elucidated but it seems plausible that it involves, at least in part, spontaneous partitioning due to the physico-chemical properties of the wax compounds and interactions with the intracuticular polymers. The arrangement of compounds probably directly influences cuticular functions.     

Micro-Raman characterisation of the R to T state transition of haemoglobin within a single living erythrocyte

We present the first recorded Raman spectra of haemoglobin in both the R and T states from within a single living erythrocyte using 632.8 nm excitation. Bands characteristic of low spin haems are observed in oxygenated and carboxylated erythrocytes at approx. 1636 (nu(10)), 1562-1565 (nu(2)), 1250-1245 cm(-1) (nu(13)) and 1226-1224 cm(-1) (nu(5)+nu(8)). The spectra of deoxygenated and methaemoglobin erythrocytes have characteristic high spin bands at approx. 1610-1606 cm(-1) (nu(10)), 1582-1580 (nu(37)), 1547-1544 (nu(11)), 1230-1220 cm(-1) (nu(13)) and 1215-1210 cm(-1) (nu(5)+nu(8)). Bands at 1172 (nu(30)), 976 (nu(45)) and 672 (nu(7)) cm(-1) appear to be enhanced at 632.8 nm in low spin haems. The oxidation state marker band (nu(4)) at 1364-1366 cm(-1) appeared invariant within this domain in all single cells and conditions investigated contrary to other resonance Raman studies on haem isolates. The information gained by in vivo single erythrocyte molecular analysis has important ramifications to the understanding of fundamental physiological processes and may have applications in the diagnosis and treatment of red blood cell disorders.     

Estimation of anisotropy coefficient of swine pancreas,liver and muscle at 1064 nm based on goniometric technique

Optical properties of tissues are required for theoretical modeling of Laser Ablation in tumor therapy. The light scattering characteristic of tissues is described by the anisotropy coefficient, g. The relationship between the angular distribution of scattered light and g is given by the Henyey‐Greenstein (HG) phase function. This work describes the estimation of anisotropy coefficients of ex vivo swine pancreas, liver and muscle at 1064 nm. The intensities of scattered light at fixed angles were measured under repeatability conditions. Experimental data were fitted with a two‐term HG, estimating the anisotropy coefficients for the forward (e.g., 0.956 for pancreas, 0.964 for liver and 0.968 for muscle) and the backward (e.g., –0.481 for pancreas, –0.414 for liver and –0.372 for muscle) scattering.

Experimental set up employed to estimate the anisotropy coefficient of biological tissues. The image on the left depicts the holder used to house tissue, laser fiber and photodetector; on the left an example of scattered light beam is shown, as well as the effect due to Snell's law.     

Structure and dynamics of the DNA-binding protein HU of B. stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy

The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a 90-residue homodimer that binds nonspecifically to B DNA. Although the structure of the HUBst:DNA complex is not known, the proposed DNA-binding surface consists of extended arms that project from an alpha-helical platform. Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface. Raman conformation markers show that the DNA-binding arms of the dimer contain beta-stranded structure in excess (eight +/- two residues per subunit) of that reported previously. Important among side-chain markers are Met (701 cm(-1)), Ala (908 cm(-1)), Arg (1082 cm(-1)), and Pro (1457 cm(-1)). The Ala marker undergoes a substantial shift (908 --> 893 cm(-1)) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines. A large subset of alanines (67%) in the alpha-helical core exhibits robust resistance to exchange. A quantitative study of NH --> ND exchange exploiting newly identified amide II' markers of helical (1440 cm(-1)) and nonhelical (1472 cm(-1)) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins.     

Cuticular waxes from potato (Solanum tuberosum) leaves

The qualitative and quantitative compositions of leaf cuticular waxes from potato (Solanum tuberosum) varieties were studied. The principal components of the waxes were very long chain n-alkanes, 2-methylalkanes and 3-methylalkanes (3.1-4.6 microg cm(-2)), primary alcohols (0.3-0.7 microg cm(-2)), fatty acids (0.3-0.6 microg cm(-2)), and wax esters (0.1-0.4 microg cm(-2)). Methyl ketones, sterols, beta-amyrin, benzoic acid esters and fatty acid methyl, ethyl, isopropyl and phenylethyl esters were found for the first time in potato waxes. The qualitative composition of the waxes was quite similar but there were quantitative differences between the varieties studied. A new group of cuticular wax constituents consisting of free 2-alkanols with odd and even numbers of carbon atoms ranging from C25 to C30 was identified.     

自然和热凝固的人肝组织对KTP/YAG激光的光学特性的比较研究

本文采用双积分球测量系统和Inverse Add ing-Doub ling方法,研究了自然和热凝固的人肝组织对532nm的KTP激光和1 064 nm的Nd:YAG激光的光学特性。结果表明:热凝固的人肝组织对532 nm的KTP激光的吸收系数较自然的肝组织的吸收系数增大了23.5%(P<0.05),热凝固的肝组织对1 064 nm的Nd:YAG激光的吸收系数较自然的肝组织的吸收系数减小了34.3%(P<0.05)。热凝固的人肝组织对532 nm的KTP激光的散射系数较自然的肝组织的散射系数增大了4.50倍(P<0.05),热凝固的肝组织对1 064 nm的Nd:YAG激光的散射系数较自然的肝组织的散射系数增大了6.41倍(P<0.05)。热凝固的人肝组织对532 nm的KTP激光的各向异性因子较自然的肝组织的各向异性因子减小了5.47%,热凝固的肝组织对1 064 nm的Nd:YAG激光的各向异性因子较自然的肝组织的各向异性因子减小了1.95%。     

Catalysis of electron transfer by selenocysteine

Selenium is an essential element that is involved in biological redox processes. The electrode potentials of the selenocysteine half-reactions RSe(*) + e(-) --> RSe-, (RSeSeR)(*)(-) + e(-) --> 2 RSe(-), and RSeSeR + 2 e(-) --> 2 RSe(-) [E degrees' (pH 7)] are +0.43, +0.18, and -0.38 V, respectively, at pH 7. The spectra of RSe(*) and (RSeSeR)(*)(-) are characterized by absorption maxima at 460 nm (epsilon = 560 M(-)(1) cm(-)(1)) and 455 nm (epsilon = 7100 M(-)(1) cm(-)(1)), respectively. The bond dissociation energy of RSe-H has been calculated, and the value of 310 kJ/mol is in agreement with literature values. In comparison with the sulfur analogue cysteine, the more facile accessibility of the radical oxidation state is striking and may have biological implications, such as in mediation of one-electron- and two-electron-transfer processes, as illustrated by catalysis by selenocysteine of the electron transfer between dithiothreitol and benzyl viologen.     

Mite-control activities of active constituents isolated from Pelargonium graveolens against house dust mites

The mite-control activities of materials obtained from Pelargonium graveolens oil against Dermatophagoides farinae and D. pteronyssinus were examined using an impregnated fabric disk bioassay and were compared with those shown by commercial benzyl benzoate and N,N-diethylm- toluamide (DEET). Purification of the biologically active constituents from P. graveolens oil was done by silica gel chromatography and high performance liquid chromatography. The structures of the active components were analyzed by EI/MS, (1)H-NMR, (13)C-NMR, (1)H-(13)C COSYNMR, and DEPT-NMR spectra, and were identified as geraniol (C(10)H(18)O, MW 154.25, trans-3,7-dimethyl-2,6- octadien-1-ol) and beta-citronellol (C(10)H(20)O, MW 156.27, 3,7-dimethyl-6-octen-1-ol). Based on the LD50 values, the most toxic compound was geraniol (0.26 microg/cm(2)), followed by beta-citronellol (0.28 microg/cm(2)), benzyl benzoate (10.03 microg/ cm(2)), and DEET (37.12 microg/cm(2)) against D. farinae. In the case of D. pteronyssinus, geraniol (0.28 microg/cm(2)) was the most toxic, followed by beta-citronellol (0.29 microg/cm(2)), benzyl benzoate (9.58 microg/cm(2)), and DEET (18.23 microg/cm(2)). These results suggest that D. farinae and D. pteronyssinus may be controlled more effectively by the application of geraniol and beta-citronellol than benzyl benzoate and DEET. Furthermore, geraniol and beta-citronellol isolated from P. graveolens could be useful for managing populations of D. farinae and D. pteronyssinus.     

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.      

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Temporal increase in the reactivity of pulmonary vasculature to substance P in chronically hypoxic rats

We previously demonstrated that the pulmonary vascular response to substance P (SP) increased in chronically hypoxic rats. This study explored the temporal increase in reactivity of the pulmonary vascular response to SP and its underlying mechanisms. First, young female Wistar rats were exposed to sea level (SL) or simulated high altitude (HA) for 15 h/day for 3 days, 1 wk, 2 wk, and 4 wk. Lungs were isolated and perfused with 4% bovine serum albumin in Krebs-Henseleit buffer solution. SP (1.5 x 10(-4) M) induced significant increases in pulmonary arterial pressure (P(pa)), venous pressure (P(v)), capillary pressure (P(c)), arterial resistance (R(a)), and filtration coefficient (K(fc)) in SL lungs. Increases in P(pa) and R(a) were significantly augmented in HA lungs, with a temporal increase trend peaking at 2 wk of HA exposure. The selective neurokinin (NK) type 1 (NK1) receptor antagonist SR-14033 significantly attenuated SP-induced increases in P(pa), P(v), P(c), R(a), and K(fc) in SL lungs. In lungs exposed to HA for 2 wk, SR-14033 suppressed the effect of SP on P(pa). Also, chronic hypoxia induced significant increases in NK1 receptors and NK1 receptor mRNA, with a temporal trend. We conclude that chronic hypoxia temporally augments SP-induced vascular responses, which are closely associated with increases in NK1 receptors and gene expression.