Reflectance spectroscopy for diagnosis of epithelial precancer: model-based analysis of fiber-optic probe designs to resolve spectral information from epithelium and stroma

Reflectance spectroscopy is a promising technology for detection of epithelial precancer. Fiber-optic probes that selectively collect scattered light from both the epithelium and the underlying stroma are likely to improve diagnostic performance of in vivo reflectance spectroscopy by revealing diagnostic features unique to each layer. We present Monte Carlo models with which to evaluate fiber-optic probe geometries with respect to sampling depth and depth resolution. We propose a probe design that utilizes half-ball lens coupled source and detector fibers to isolate epithelial scattering from stromal scattering and hence to resolve spectral information from the two layers. The probe is extremely compact and can provide easy access to different organ sites.     

Solvent-assisted patterning of polyelectrolyte multilayers and selective deposition of virus assemblies

We introduce a simple method to pattern electrostatic assemblies of viruses onto a polyelectrolyte multilayer. The increased mobility of weak polycation chains in the multilayer above a given thickness ensures the surface mobility of viruses required for spontaneous ordering of densely packed viruses atop polymeric patterns. To pattern the polyelectrolyte multilayer film, we employ a nonconventional patterning method known as solvent-assisted capillary molding for the first time on multilayer films, and demonstrate micrometer-scaled dense patterns of viruses, where the accessible feature size can be correlated by the length scale of virus and swelling property of underlying patterned polyelectrolyte multilayer. We further examine the ability to modify the top surfaces of these assemblies with biological ligands, which extends the applicability of patterned viruses to biological detection purposes. We expect that the present method described here can be generally applied to the patterning of other polyelectrolyte multilayers and combined with the ordered assembly of anisotropic nanomaterials such as polymeric nanotubes or inorganic nanowires for a broad range of applications.     

Light scattering of ultrafine silica particles by VUV synchrotron radiation

Vacuum ultraviolet (VUV) light scattering from ultrafine silica particles is studied with an aerosol instrument recently established at the Advanced Light Source (ALS) in Berkeley. Silica particles, size-selected by a differential mobility analyzer, are introduced into vacuum through a set of aerodynamic lenses to form a particle beam. The scattered photons from the crossing area of the VUV synchrotron beam and particle beam are detected with a rotatable VUV photon detector. The angular distributions of scattered photons (ADSP) originating from 70, 100, 200 nm diameter silica particles are measured with 145.9 and 118.1 nm synchrotron radiation. These angular distributions show strong forward scattering. The measured ADSPs are consistent with simulation of Mie scattering. The refractive indices of silica particles, 2.6 + 1.1i and 1.6 + 0.0001i for 118.1 and 145.9 nm, respectively, are obtained by fitting the measured ADSPs; the least average percentage deviations are 18% and 6%, respectively. The scattered fluxes at widely different wavelengths (visible versus VUV) also exhibit clear size sensitivity. Under comparable experimental conditions of photon fluxes and detection efficiencies, limits of particle size detection of 70 and 250 nm are obtained, respectively, when using 118.1 and 532 nm illumination. As anticipated, VUV scattering is a more sensitive probe for ultrafine particles, which will find application in detection of these ubiquitous species beyond the confines of a laboratory.     

Biosynthesis of silver nanoparticles in collagen gel improves their medical use in periodontitis treatment

Silver nanoparticles (AgNP) suspensions were biosynthesized by silver ions reduction in the presence of collagen, a nontoxic, organic polymer, intending to improve their medical use in periodontitis treatment. Spectrophotometric measurements showed a time- and concentration-dependent increase of AgNP formation in each suspension variant. Transmission electron microscopy revealed spherical morphology of AgNP in collagen and their mean diameter size was around 30?nm. The particle size distribution and zeta potential values of AgNP in collagen were determined by dynamic light scattering measurements. The surface charge of AgNP in collagen was positive, while commercial AgNP stabilized in citrate had negative surface charge. In vitro cytotoxicity testing of AgNP in collagen showed that they were biocompatible with human gingival fibroblasts in a wider range of concentrations than commercial nanoparticles. The antibacterial activity of AgNP in collagen against two pathogenic strains present in the periodontal pocket was dose-dependent and higher than that of AgNP in citrate. All these results demonstrated that AgNP prepared in collagen gel had improved properties, like small diameter, positive surface charge, high biocompatibility in human gingival fibroblasts, efficiency against bacterial growth and, thus, better therapeutic potential in periodontal disease treatment.     

Measurement of particle size distribution in mammalian cells in vitro by use of polarized light spectroscopy

We demonstrate the feasibility of measuring the particle size distribution (PSD) of internal cell structures in vitro. We use polarized light spectroscopy to probe the internal morphology of mammalian breast cancer (MCF7) and cervical cancer (Siha) cells. We find that graphing the least-squared error versus the scatterer size provides insight into cell scattering. A nonlinear optimization scheme is used to determine the PSD iteratively. The results suggest that 2-microm particles (possibly the mitochondria) contribute most to the scattering. Other subcellular structures, such as the nucleoli and the nucleus, may also contribute significantly. We reconstruct the PSD of the mitochondria, as verified by optical microscopy. We also demonstrate the angle dependence of the PSD.     

Estimating and correcting mie scattering in synchrotron-based microscopic fourier transform infrared spectra by extended multiplicative signal correction

We present an approach for estimating and correcting Mie scattering occurring in infrared spectra of single cells, at diffraction limited probe size, as in synchrotron based microscopy. The Mie scattering is modeled by extended multiplicative signal correction (EMSC) and subtracted from the vibrational absorption. Because the Mie scattering depends non-linearly on alpha, the product of the radius and the refractive index of the medium/sphere causing it, a new method was developed for estimating the Mie scattering by EMSC for unknown radius and refractive index of the Mie scatterer. The theoretically expected Mie contributions for a range of different alpha values were computed according to the formulae developed by Van de Hulst (1957). The many simulated spectra were then summarized by a six-dimensional subspace model by principal component analysis (PCA). This subspace model was used in EMSC to estimate and correct for Mie scattering, as well as other additive and multiplicative interference effects. The approach was applied to a set of Fourier transform infrared (FT-IR) absorbance spectra measured for individual lung cancer cells in order to remove unwanted interferences and to estimate ranges of important alpha values for each spectrum. The results indicate that several cell components may contribute to the Mie scattering.     

Optical probes for biological applications based on surface-enhanced Raman scattering from indocyanine green on gold nanoparticles

We report surface-enhanced Raman scattering (SERS) studies on indocyanine green (ICG) on colloidal silver and gold and demonstrate a novel optical probe for applications in living cells. In addition to its own detection by the characteristic ICG SERS signatures, the ICG gold nanoprobe delivers spatially localized chemical information from its biological environment by employing SERS in the local optical fields of the gold nanoparticles. The probe offers the potential to increase the spectral specificity and selectivity of current chemical characterization approaches of living cells and biomaterials based on vibrational information.     

Particle-size and velocity measurements in flowing conditions using dynamic light scattering

The noninvasive optical technique of dynamic light scattering (DLS) is routinely used to characterize dilute and transparent submicrometer particle dispersions in laboratory environments. A variety of industrial and biological applications would, however, greatly benefit from on-line monitoring of dispersions under flowing conditions. We present a model experiment to study flowing dispersions of polystyrene latex particles of varying sizes under varying flow conditions by using a newly developed fiber-optic DLS probe. A modified correlation function proposed in an earlier study is applied to the analysis of extracting the size and velocity of laminar flowing particulate dispersions. The complementary technique of laser Doppler velocimetry is also used to measure the speed of moving particles to confirm the DLS findings.     

Surface imaging using holographic optical tweezers

We present an imaging technique using an optically trapped cigar-shaped probe controlled using holographic optical tweezers. The probe is raster scanned over a surface, allowing an image to be taken in a manner analogous to scanning probe microscopy (SPM), with automatic closed loop feedback control provided by analysis of the probe position recorded using a high speed CMOS camera. The probe is held using two optical traps centred at least 10 μm from the ends, minimizing laser illumination of the tip, so reducing the chance of optical damage to delicate samples. The technique imparts less force on samples than contact SPM techniques, and allows highly curved and strongly scattering samples to be imaged, which present difficulties for imaging using photonic force microscopy. To calibrate our technique, we first image a known sample--the interface between two 8 μm polystyrene beads. We then demonstrate the advantages of this technique by imaging the surface of the soft alga Pseudopediastrum. The scattering force of our laser applied directly onto this sample is enough to remove it from the surface, but we can use our technique to image the algal surface with minimal disruption while it is alive, not adhered and in physiological conditions. The resolution is currently equivalent to confocal microscopy, but as our technique is not diffraction limited, there is scope for significant improvement by reducing the tip diameter and limiting the thermal motion of the probe.     

Nanomaterials for X-ray imaging: gold nanoparticle enhancement of X-ray scatter imaging of hepatocellular carcinoma

We present the development of a new imaging technique for the early diagnosis of hepatocellular carcinoma that utilizes surface-modified gold nanoparticles in combination with X-ray imaging. Tissues labeled with these electron-dense particles show enhanced X-ray scattering over normal tissues, distinguishing cells containing gold nanoparticles from cells without gold in X-ray scatter images. Our results suggest that this novel approach could enable the in vivo detection of tumors as small as a few millimeters in size.     

Liquid chromatographic arsenic speciation with gas-phase chemiluminescence detection

We present a newly developed gas-phase chemiluminescence (CL) detection method for the separation and quantification of inorganic and organic arsenic species. Arsenite, arsenate, dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA) were separated by anion exchange using carbonate-bicarbonate and NaOH eluents with step-gradient elution. The separated species were passed through a UV photooxidation reactor which decomposed the organic species and converted them to inorganic As(V). Subsequent on-line hydride generation with acid and sodium borohydride produces AsH3 and H2, which are separated from the liquid in a gas-liquid separator. The produced AsH3, driven by H2, reacts with ozone in a small reflective cell located atop a photomultiplier tube, resulting in intense CL. In the present form, the limits of detection (LODs, signal-to-noise = 3), based on peak height, for arsenite, arsenate, MMA, and DMA are 0.4, 0.2, 0.5, and 0.3 microg/L, respectively, for a 100 microL injected sample. This analyzer demonstrates the robustness of the CL detection system for arsenic and provides an affordable alternative to atomic spectrometry for use as a detector after chromatographic speciation. We found no significant practical interferences.     

Measured Infrared Spectral Extinction for Aerosolized Bacillus subtilis var. niger Endospores from 3 to 13 mum

We measured spectral extinction in situ for aerosolized Bacillus subtilis var. niger endospores using Fourier-transform infrared spectroscopy from 3.0 to 13.0 mum. Corresponding aerosol size distributions were measured with a commercially available elastic light-scattering probe and verified by direct particle capture and subsequent counting by video microscopy. Aerosol mass density was monitored simultaneously with conventional dosimetry and was used to mass normalize the measured spectral extinction. Mie theory calculations based on measured distributions and available complex indices of refraction agreed well. We also present resultant Mie calculations for the absorption, total scattering, and backscatter. For comparison, measured spectral extinction for three common environmental aerosols is also presented, i.e., for water fog, diesel soot, and Arizona road dust.     

NMR analysis on microfluidic devices by remote detection

We present a novel approach to perform high-sensitivity NMR imaging and spectroscopic analysis on microfluidic devices. The application of NMR, the most information-rich spectroscopic technique, to microfluidic devices remains a challenge because the inherently low sensitivity of NMR is aggravated by small fluid volumes leading to low NMR signal and geometric constraints resulting in poor efficiency for inductive detection. We address the latter by physically separating signal detection from encoding of information with remote detection. Thereby, we use a commercial imaging probe with sufficiently large diameter to encompass the entire device, enabling encoding of NMR information at any location on the chip. Because large-diameter coils are too insensitive for detection, we store the encoded information as longitudinal magnetization and flow it into the outlet capillary. There, we detect the signal with optimal sensitivity, using a solenoidal microcoil, and reconstruct the information encoded in the fluid. We present a generally applicable design for a detection-only microcoil probe that can be inserted into the bore of a commercial imaging probe. Using hyperpolarized 129Xe gas, we show that this probe enables sensitive reconstruction of NMR spectroscopic information encoded by the large imaging probe while keeping the flexibility of a large coil.     

Polarized X-ray scattering reveals non-crystalline orientational ordering in organic films

Molecular orientation critically influences the mechanical, chemical, optical and electronic properties of organic materials. So far, molecular-scale ordering in soft matter could be characterized with X-ray or electron microscopy techniques only if the sample exhibited sufficient crystallinity. Here, we show that the resonant scattering of polarized soft X-rays (P-SoXS) by molecular orbitals is not limited by crystallinity and that it can be used to probe molecular orientation down to size scales of 10 nm. We first apply the technique on highly crystalline small-molecule thin films and subsequently use its high sensitivity to probe the impact of liquid-crystalline ordering on charge mobility in polymeric transistors. P-SoXS also reveals scattering anisotropy in amorphous domains of all-polymer organic solar cells where interfacial interactions pattern orientational alignment in the matrix phase, which probably plays an important role in the photophysics. The energy and q-dependence of the scattering anisotropy allows the identification of the composition and the degree of orientational order in the domains.     

跑道型差动式涡流探头设计及其性能研究

针对传统差动式涡流探头几何尺寸大、缺陷检测灵敏度低的问题,在传统差动式涡流探头的基础上,设计了一种跑道型差动式涡流探头。它由激励线圈包裹2个尺寸相同、反向连接的感应线圈而构成。首先,利用COMSOL Multiphysics仿真软件建立了跑道型差动式涡流探头模型,比较了跑道型差动式涡流探头与传统差动式涡流探头涡流场分布的差异,并研究了在不同缺陷深度、不同扫查角度下2种差动式涡流探头的检测灵敏度。接着,制作了跑道型差动式涡流探头实物和碳钢板缺陷试件,利用试验测试的方法比较了跑道型和传统差动式涡流探头的检测灵敏度。试验结果表明,与传统差动式涡流探头相比,跑道型差动式涡流探头具有更紧凑的结构、更高的缺陷检测灵敏度。研究结果可为小尺寸、高精度差动式涡流探头的优化设计提供参考。     

Elastic light-scattering measurements of single biological cells in an optical trap

We have developed an instrument for determination of the angular light scattering of beads and biological cells. The instrument uses radiation pressure for levitation of particles inside a cuvette. The setup consists of two 780-nm diode lasers in a vertical double-beam trapping configuration. In the horizontal direction a weakly focused 633-nm probe beam is used to illuminate the trapped particle. One can detect scattered light over the range of from - 150 to 150 deg with an angular resolution of 0.9 deg using an avalanche photodiode. With this setup light scattering from polystyrene beads was measured, and the obtained scattering patterns were compared with theoretical scattering patterns from Lorenz-Mie theory. The results show that the setup is stable, gives reproducible patterns, and qualitatively agrees with the calculations. Trapping of biological cells is more difficult than trapping of beads, because smaller forces result from smaller refractive indices. We present an angular scattering pattern measured from a human lymphocyte measured from 20 to 60 deg.     

Null detection of ballistic photons for locating objects buried in turbid media

We describe an optical system in which a vibration induced to a laser probe beam combined with a half-blocked photodiode allows determination of the position and width of objects buried in turbid media. Our system is based on the detection of an AC signal which drastically decreases under the presence of an obstructing buried object. We describe the technique and include experimental results showing that the system is capable of detecting 2?mm wide objects buried at depths up to 3?cm from the front surface of a sample simulating scattering properties of soft tissue.     

Measuring the optical parameters of weakly absorbing, highly turbid suspensions by a new technique: photoacoustic detection of scattered light

We present and apply a novel method, the scattering photoacoustic (SPA) technique, for measuring optical parameters in weakly absorbing, highly scattering suspensions. In this method, a solid absorber is in contact with a suspension sample to permit the photoacoustic detection of the sample's light-scattering properties. We conducted measurements conducted to determine the reduced scattering coefficients of Intralipid suspensions with a concentration range of 0.1-5%, and the results are in good agreement with those achieved by other researchers. Moreover, we also illustrate the relationship between the amplitude of the SPA signal and absorption, scattering, and detection distance. Through a study of Intralipid-ink mixes, we demonstrate that the SPA technique has the ability to determine simultaneously the absorption and reduced scattering coefficients of turbid media. This new technique has low cost and is noninvasive, and it enables on-line measurements to be made.     

Doppler encoded excitation pattern tomographic optical microscopy

Most far-field optical imaging systems rely on lenses and spatially resolved detection to probe distinct locations on the object. We describe and demonstrate a high-speed wide-field approach to imaging that instead measures the complex spatial Fourier transform of the object by detecting its spatially integrated response to dynamic acousto-optically synthesized structured illumination. Tomographic filtered backprojection is applied to reconstruct the object in two or three dimensions. This technique decouples depth of field and working distance from resolution, in contrast to conventional imaging, and can be used to image biological and synthetic structures in fluoresced or scattered light employing coherent or broadband illumination. We discuss the electronically programmable transfer function of the optical system and its implications for imaging dynamic processes. We also explore wide-field fluorescence imaging in scattering media by coherence gating. Finally, we present two-dimensional high-resolution tomographic image reconstructions in both scattered and fluoresced light demonstrating a thousandfold improvement in the depth of field compared to conventional lens-based microscopy.     

Tip-enhanced Raman detection of antibody conjugated nanoparticles on cellular membranes

Tip enhanced Raman scattering (TERS) microscopy is used to image antibody conjugated nanoparticles on intact cellular membranes. The combination of plasmonic coupling and the resultant electric field obtained from intermediate focusing of a radially polarized source gives rise to Raman images with spatial resolution below 50 nm. Finite element method calculations are used to explain the origins of the observed image resolution and spectroscopic signals. The observed Raman scattering provides information about the biomolecules present near the nanoparticle probes. The results show that aggregates of nanoparticles produce spectroscopic results similar to those reported from other surface enhanced Raman spectroscopies, e.g., shell isolated nanoparticle enhanced Raman spectroscopy (SHINERS) and aggregated nanoparticles; however, TERS enables the detection of isolated nanoparticles on cell membranes where the observed spectra provide information about the interaction of the specific biomolecule conjugated to the nanoparticle probe. These measurements present a new technique for exploring biomolecular interactions on the surface of cells and tissue.