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The technique of phase contrast imaging, combined with tomographic reconstructions, can rapidly measure ultrasonic fields propagating in water, including ultrasonic fields with complex wavefront shapes, which are difficult to characterize with standard hydrophone measurements. Furthermore, the technique can measure the absolute pressure amplitudes of ultrasonic fields without requiring a pressure calibration. Absolute pressure measurements have been previously demonstrated using optical imaging methods for ultrasonic frequencies below 2.5 MHz. The present work demonstrates that phase contrast imaging can accurately measure ultrasonic fields with frequencies up to 20 MHz and pressure amplitudes near 10 kPa. Accurate measurements at high ultrasonic frequencies are performed by tailoring the measurement conditions to limit optical diffraction as guided by a simple dimensionless parameter. In some situations, differences between high frequency measurements made with the phase contrast method and a calibrated hydrophone become apparent, and the reasons for these differences are discussed. Extending optical imaging measurements to high ultrasonic frequencies could facilitate quantitative applications of ultrasound measurements in nondestructive testing and medical therapeutics and diagnostics such as photoacoustic imaging.
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The measurement of the spatial distribution of oxygen saturation (sO2) in superficial tissues using optical reflectance imaging has been useful in the clinical venue especially in temporally demanding applications such as monitoring tissue oxygenation during surgery. The measurement is based on relative spectrometry of oxy- and deoxyhemoglobin in tissues. We titrated deoxyhemoglobin with oxygen gas and simultaneously measured the dissolved oxygen pressure and the visible absorbance spectra to verify spectral shapes at different saturations. sO2 values derived from the measured pO2 are compared to those derived from the hemoglobin spectra at various stages of oxygenation.
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Oxígeno/análisis , Oxihemoglobinas/análisis , Calibración , Humanos , Presión Parcial , SolucionesRESUMEN
We report on an experimental procedure in confocal single molecule fluorescence lifetime correlation spectroscopy (FLCS) to determine the range of excitation power and molecular or particulate concentration in solution under which the application of an unmodified model autocorrelation function is justified. This procedure enables fitting of the autocorrelation to an accurate model to measure diffusion length (r) and diffusion time (τD) of single molecules in solution. We also report on the pinhole size dependency of r and τD in a confocal FLCS platform. This procedure determines a set of experimental parameters with which the Stokes-Einstein (S-E) equation accurately measures the hydrodynamic radii of spherical nanoparticles, enabling the determination of the particle size range for which the hydrodynamic radius by the S-E equation measures the real particle radius.
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Significance: Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries. Aim: We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples. Approach: Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the K-means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes. Results: The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised K-means algorithm. The unsupervised K-means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within <2% residual error margin. Conclusions: Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to <2% residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.
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Algoritmos , Neoplasias de la Mama , Mastectomía Segmentaria , Microscopía , Humanos , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Neoplasias de la Mama/patología , Femenino , Mastectomía Segmentaria/métodos , Microscopía/métodos , Mama/diagnóstico por imagen , Mama/patología , Mama/cirugía , Imágenes Hiperespectrales/métodos , Márgenes de Escisión , Método de Montecarlo , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3G (A3G) is a cytidine deaminase that restricts retroviruses, endogenous retro-elements and DNA viruses. A3G plays a key role in the anti-HIV-1 innate cellular immunity. The HIV-1 Vif protein counteracts A3G mainly by leading A3G towards the proteosomal machinery and by direct inhibition of its enzymatic activity. Both activities involve direct interaction between Vif and A3G. Disrupting the interaction between A3G and Vif may rescue A3G antiviral activity and inhibit HIV-1 propagation. Here, mapping the interaction sites between A3G and Vif by peptide array screening revealed distinct regions in Vif important for A3G binding, including the N-terminal domain (NTD), C-terminal domain (CTD) and residues 83-99. The Vif-binding sites in A3G included 12 different peptides that showed strong binding to either full-length Vif, Vif CTD or both. Sequence similarity was found between Vif-binding peptides from the A3G CTD and NTD. A3G peptides were synthesized and tested for their ability to counteract Vif action. A3G 211-225 inhibited HIV-1 replication in cell culture and impaired Vif dependent A3G degradation. In vivo co-localization of full-length Vif with A3G 211-225 was demonstrated by use of FRET. This peptide has the potential to serve as an anti-HIV-1 lead compound. Our results suggest a complex interaction between Vif and A3G that is mediated by discontinuous binding regions with different affinities.
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Fármacos Anti-VIH/química , Citidina Desaminasa/química , Mapeo Peptídico , Péptidos/química , Análisis por Matrices de Proteínas , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Desaminasa APOBEC-3G , Células Cultivadas , Citidina Desaminasa/aislamiento & purificación , Citidina Desaminasa/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Péptidos/síntesis química , Péptidos/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The determination of the level of oxygenation in optically accessible tissues using multispectral or hyperspectral imaging (HSI) of oxy- and deoxyhemoglobin has special appeal in clinical work due to its noninvasiveness, ease of use, and capability of providing molecular and anatomical information at near video rates during surgery. In this paper we refer to an example of the use of HSI in monitoring oxygenation of kidneys during partial nephrectomy. In a study using porcine models, it was found that artery-only clamping left the kidney better oxygenated, as opposed to simultaneously clamping the artery and the vein. A subsequent study correlates gradations in blood flow by partial clamping during the surgical procedure with postoperative renal function via assessment of creatinine level. We discuss the various contributions to the uncertainty of the oxygen saturation measured by this remote-sensing imaging technique in medical application.
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Isquemia/patología , Riñón/irrigación sanguínea , Riñón/patología , Nefrectomía , Oxígeno/sangre , Análisis Espectral , Animales , Calibración , Constricción , Creatinina/sangre , Hemoglobinas/metabolismo , Isquemia/cirugía , Riñón/cirugía , Arteria Renal/patología , Arteria Renal/cirugía , PorcinosRESUMEN
SIGNIFICANCE: The polymer, polydimethylsiloxane (PDMS), has been increasingly used to make tissue simulating phantoms due to its excellent processability, durability, flexibility, and limited tunability of optical, mechanical, and thermal properties. We report on a robust technique to fabricate PDMS-based tissue-mimicking phantoms where the broad range of scattering and absorption properties are independently adjustable in the visible- to near-infrared wavelength range from 500 to 850 nm. We also report on an analysis method to concisely quantify the phantoms' broadband characteristics with four parameters. AIM: We report on techniques to manufacture and characterize solid tissue-mimicking phantoms of PDMS polymers. Tunability of the absorption (µa ( λ ) ) and reduced scattering coefficient spectra (µs'(λ)) in the wavelength range of 500 to 850 nm is demonstrated by adjusting the concentrations of light absorbing carbon black powder (CBP) and light scattering titanium dioxide powder (TDP) added into the PDMS base material. APPROACH: The µa ( λ ) and µs'(λ) of the phantoms were obtained through measurements with a broadband integrating sphere system and by applying an inverse adding doubling algorithm. Analyses of µa ( λ ) and µs'(λ) of the phantoms, by fitting them to linear and power law functions, respectively, demonstrate that independent control of µa ( λ ) and µs'(λ) is possible by systematically varying the concentrations of CBP and TDP. RESULTS: Our technique quantifies the phantoms with four simple fitting parameters enabling a concise tabulation of their broadband optical properties as well as comparisons to the optical properties of biological tissues. We demonstrate that, to a limited extent, the scattering properties of our phantoms mimic those of human tissues of various types. A possible way to overcome this limitation is demonstrated with phantoms that incorporate polystyrene microbead scatterers. CONCLUSIONS: Our manufacturing and analysis techniques may further promote the application of PDMS-based tissue-mimicking phantoms and may enable robust quality control and quality checks of the phantoms.
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Dimetilpolisiloxanos , Humanos , Fantasmas de ImagenRESUMEN
Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called "NICE" (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods.
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Recuento de Colonia Microbiana/economía , Recuento de Colonia Microbiana/métodos , Escherichia coli/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/economía , Ensayos Analíticos de Alto Rendimiento/métodos , Automatización , Recuento de Colonia Microbiana/instrumentación , Escherichia coli/citología , Ensayos Analíticos de Alto Rendimiento/instrumentación , Reproducibilidad de los Resultados , Sales de Tetrazolio/metabolismoRESUMEN
Blinking of zinc blende CdSe-based core/shell nanocrystals is studied as a function of shell materials and surface ligands. CdSe/ZnS, CdSe/ZnSe/ZnS and CdSe/CdS/ZnS core/shell nanocrystals are prepared by colloidal synthesis and six monolayers of larger bandgap shell materials are grown over the CdSe core. Organic-soluble nanocrystals covered with stearate are made water-soluble by ligand exchange with 3-mercaptopropionic acid. The light-emitting states of nanocrystals are characterized by absorption and emission spectroscopy as well as photoluminescence lifetime measurements in solution. The blinking time trace is recorded for single nanocrystals on a glass coverslip. Both on- and off-time distributions are fitted to the power law. The power-law exponents vary, depending on shell materials and surface ligands. The off-time exponents for organic and water-soluble nanocrystals are measured in the range of 1.36-1.55 and 1.25-1.37, respectively, while their on-time exponents are spread in the range of 1.53-1.86 and 1.85-2.17, respectively. Water-soluble surface passivation with thiolate prolongs the dark period regardless of shell materials and core/shell structures. Of the core/shell structures, CdSe/CdS/ZnS exhibits the longest bright state. The on/off-time exponents are inversely correlated, although the successive on/off events are not individually correlated. A two competing charge-tunneling model is presented to describe the variation of on- and off-time exponents with shell materials and surface ligands.
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Compuestos de Cadmio/química , Ligandos , Nanopartículas/química , Compuestos de Selenio/química , Sulfuros/química , Termodinámica , Compuestos de Zinc/químicaRESUMEN
We report the development of an optically transparent high-frequency ultrasonic transducer using lithium niobate single-crystal and indium-tin-oxide electrodes with up to 90% optical transmission in the visible-to-near-infrared spectrum. The center frequency of the transducer was at 36.9 MHz with 33.9%, at -6 dB fractional bandwidth. The photoacoustic imaging capability of the fabricated transducer was also demonstrated by successfully imaging a resolution target and mouse-ear vasculatures in vivo, which were irradiated by a 532 nm pulse laser transmitted through the transducer.
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Microscopía Acústica/instrumentación , Técnicas Fotoacústicas/instrumentación , Transductores , Animales , Oído/irrigación sanguínea , Oído/diagnóstico por imagen , Diseño de Equipo , Ratones , Fantasmas de ImagenRESUMEN
Spectrally resolved photoacoustic imaging is promising for label-free imaging in optically scattering materials. However, this technique often requires acquisition of a separate image at each wavelength of interest. This reduces imaging speeds and causes errors if the sample changes in time between images acquired at different wavelengths. We demonstrate a solution to this problem by using dual-comb spectroscopy for photoacoustic measurements. This approach enables a photoacoustic measurement at thousands of wavelengths simultaneously. In this technique, two optical-frequency combs are interfered on a sample and the resulting pressure wave is measured with an ultrasound transducer. This acoustic signal is processed in the frequency-domain to obtain an optical absorption spectrum. For a proof-of-concept demonstration, we measure photoacoustic signals from polymer films. The absorption spectra obtained from these measurements agree with those measured using a spectrophotometer. Improving the signal-to-noise ratio of the dual-comb photoacoustic spectrometer could enable high-speed spectrally resolved photoacoustic imaging.
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We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (lambda) phage, and Escherichia coli are examined by several cross-validated methods. Flow and image-based cytometry clarify fluorescent labeling of bacteria, with image-based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image-based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4-17 QDs) and show that lambda phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal-biological conjugates.
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Bacteriófago lambda/metabolismo , Bacteriófago lambda/fisiología , Escherichia coli/aislamiento & purificación , Puntos Cuánticos , Coloración y Etiquetado/métodos , Acoplamiento Viral , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de la Cápside/metabolismo , Escherichia coli/virología , Citometría de Flujo , Fluorescencia , Microscopía Electrónica de Transmisión , Datos de Secuencia MolecularRESUMEN
Mid-infrared (MIR) microscopy provides rich chemical and structural information about biological samples, without staining. Conventionally, the long MIR wavelength severely limits the lateral resolution owing to optical diffraction; moreover, the strong MIR absorption of water ubiquitous in fresh biological samples results in high background and low contrast. To overcome these limitations, we propose a method that employs photoacoustic detection highly localized with a pulsed ultraviolet (UV) laser on the basis of the Grüneisen relaxation effect. For cultured cells, our method achieves water-background suppressed MIR imaging of lipids and proteins at UV resolution, at least an order of magnitude finer than the MIR diffraction limits. Label-free histology using this method is also demonstrated in thick brain slices. Our approach provides convenient high-resolution and high-contrast MIR imaging, which can benefit diagnosis of fresh biological samples.
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Digital optical phase conjugation (DOPC) enables many optical applications by permitting focusing of light through scattering media. However, DOPC systems require precise alignment of all optical components, particularly of the spatial light modulator (SLM) and camera, in order to accurately record the wavefront and perform playback through the use of time-reversal symmetry. We present a digital compensation technique to optimize the alignment of the SLM in five degrees of freedom, permitting focusing through thick scattering media with a thickness of 5 mm and transport scattering coefficient of 2.5 mm - 1 while simultaneously improving focal quality, as quantified by the peak-to-background ratio, by several orders of magnitude over an unoptimized alignment.
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Procesamiento de Imagen Asistido por Computador/métodos , Imagen Óptica/métodos , Dispositivos Ópticos , Dispersión de RadiaciónRESUMEN
We present broadband measurements of the optical properties of tissue-mimicking solid phantoms using a single integrating sphere to measure the hemispherical reflectance and transmittance under a direct illumination at the normal incident angle. These measurements are traceable to reflectance and transmittance scales. An inversion routine using the output of the adding-doubling algorithm restricted to the reflectance and transmittance under a direct illumination was developed to produce the optical parameters of the sample along with an uncertainty budget at each wavelength. The results for two types of phantoms are compared to measurements by time-resolved approaches. The results between our method and these independent measurements agree within the estimated measurement uncertainties.
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A new optical scattering contrast-agent based on polymer-nanoparticle encapsulated silver nanoplates (PESNs) is presented. Silver nanoplates were chosen due to the flexibility of tuning their plasmon frequencies. The polymer coating preserves their physical and optical properties and confers other advantages such as controlled contrast agent delivery. Finite difference time domain (FDTD) simulations model the interaction of light with the nanoplates in different orientations in the cluster. Hyperspectral dark field microscopy (HYDFM) observes the scattering spectra of the PESNs. An unsupervised sequential maximum angle convex cone (SMACC) image analysis resolves spectral endmembers corresponding to different stacking orientations of the nanoplates. The orientation-dependent endmembers qualitatively agree with the FDTD results. For contrast enhancement, the uptake and spatial distribution of PESNs are demonstrated by an HYDFM study of single melanoma cells to result in an enhanced contrast of up to 400%. A supervised spatial mapping of the endmembers obtained by the unsupervised SMACC algorithm reveals spatial distributions of PESNs with various clustering orientations of encapsulated nanoplates. Our study demonstrates tunability in plasmonics properties in clustered metal nanoparticles and its utility for the development of scatter-based imaging contrast agents for a broad range of applications, including studies of single cells and other biomedical systems.
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Nanopartículas del Metal , Microscopía , Polímeros , Plata , LuzRESUMEN
The National Institute of Standards and Technology (NIST) has maintained scales for reflectance and transmittance over several decades. The scales are primarily intended for regular transmittance, mirrors, and solid surface scattering diffusers. The rapidly growing area of optical medical imaging needs a scale for volume scattering of diffuse materials that are used to mimic the optical properties of tissue. Such materials are used as phantoms to evaluate and validate instruments under development intended for clinical use. To address this need, a double-integrating sphere based instrument has been installed to measure the optical properties of tissue-mimicking phantoms. The basic system and methods have been described in previous papers. An important attribute in establishing a viable calibration service is the estimation of measurement uncertainties. The use of custom models and comparisons with other established scales enabled uncertainty measurements. Here, we describe the continuation of those efforts to advance the understanding of the uncertainties through two independent measurements: the bidirectional reflectance distribution function and the bidirectional transmittance distribution function of a commercially available solid biomedical phantom. A Monte Carlo-based model is used and the resulting optical properties are compared to the values provided by the phantom manufacturer.
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Localized measurements of scattering in biological tissue provide sensitivity to microstructural morphology but have limited utility to wide-field applications, such as surgical guidance. This study introduces sub-diffusive spatial frequency domain imaging (sd-SFDI), which uses high spatial frequency illumination to achieve wide-field sampling of localized reflectances. Model-based inversion recovers macroscopic variations in the reduced scattering coefficient [Formula: see text] and the phase function backscatter parameter (γ). Measurements in optical phantoms show quantitative imaging of user-tuned phase-function-based contrast with accurate decoupling of parameters that define both the density and the size-scale distribution of scatterers. Measurements of fresh ex vivo breast tissue samples revealed, for the first time, unique clustering of sub-diffusive scattering properties for different tissue types. The results support that sd-SFDI provides maps of microscopic structural biomarkers that cannot be obtained with diffuse wide-field imaging and characterizes spatial variations not resolved by point-based optical sampling.
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There is a need for a common reference point that will allow for the comparison of the optical properties of tissue-mimicking phantoms. After a brief review of the methods that have been used to measure the phantoms for a contextual backdrop to our approach, this paper reports on the establishment of a standardized double-integrating-sphere platform to measure absorption and reduced scattering coefficients of tissue-mimicking biomedical phantoms. The platform implements a user-friendly graphical user interface in which variations of experimental configurations and model-based analysis are implemented to compute the coefficients based on a modified inverse adding-doubling algorithm allowing a complete uncertainty evaluation. Repeatability and validation of the measurement results of solid phantoms are demonstrated for three samples of different thicknesses, d = 5.08 mm, 7.09 mm, and 9.92 mm, with an absolute error estimate of 4.0% to 5.0% for the absorption coefficient and 11% to 12% for the reduced scattering coefficient (k = 2). The results are in accordance with those provided by the manufacturer. Measurements with different polarization angles of the incident light are also presented, and the resulting optical properties were determined to be equivalent within the estimated uncertainties.
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Diagnóstico por Imagen/normas , Óptica y Fotónica/normas , Fantasmas de Imagen/normas , Algoritmos , Anisotropía , Calibración , Humanos , Rayos Láser , Refractometría , Reproducibilidad de los Resultados , Dispersión de Radiación , Estados UnidosRESUMEN
A hyperspectral image projector (HIP) based on liquid crystal on silicon spatial light modulators is explained and demonstrated to generate data cubes. The HIP-constructed data cubes are three-dimensional images of the spatial distribution of spectrally resolved abundances of intracellular light-absorbing oxyhemoglobin molecules in single erythrocytes. Spectrally and spatially resolved image data indistinguishable from the real scene may be used as standard data cubes, so-called digital phantoms, to calibrate image sensors and validate image analysis algorithms for their measurement quality, performance consistency, and interlaboratory comparisons for quantitative biomedical imaging applications.