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Angularly resolved light scattering (ALS) has become a useful tool for assessing the size and refractive index of biological scatterers at cellular and organelle length scales. Sizing organelle populations with ALS relies on Mie scattering theory models, which require significant assumptions about the object, including spherical scatterers and a homogeneous medium. These assumptions may incur greater error at the single cell level, where there are fewer scatterers to be averaged over. We investigate the validity of these assumptions using 3D refractive index (RI) tomograms measured via optical diffraction tomography (ODT). We compute the angular scattering on digitally manipulated tomograms with increasingly strong model assumptions, including RI-matched immersion media, homogeneous cytosol, and spherical organelles. We also compare the tomogram-computed angular scattering to experimental measurements of angular scattering from the same cells to ensure that the ODT-based approach accurately models angular scattering. We show that enforced RI-matching with the immersion medium and a homogeneous cytosol significantly affects the angular scattering intensity shape, suggesting that these assumptions can reduce the accuracy of size distribution estimates.
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While osteoporosis is reliably diagnosed using dual energy X-ray absorptiometry (DXA), screening rates are alarmingly low, contributing to preventable fractures. Raman spectroscopy (RS) can detect biochemical changes that occur in bones transcutaneously and can arguably be more accessible than DXA as a fracture risk assessment. A reasonable approach to translate RS is to interrogate phalangeal bones of human hands, where the soft tissues covering the bone are less likely to hamper transcutaneous measurements. To that end, we set out to first determine whether Raman spectra obtained from phalangeal bones correlate with distal radius fracture strength, which can predict subsequent osteoporotic fractures at the spine and hip. We performed RS upon diaphyseal and epiphyseal regions of exposed proximal phalanges from 12 cadaver forearms classified as healthy (n = 3), osteopenic (n = 4), or osteoporotic (n = 5) based on wrist T-scores measured by DXA. We observed a significant decrease in phosphate to matrix ratio and a significant increase in carbonate substitution in the osteoporotic phalanges relative to healthy and osteopenic phalanges. Multivariate regression models produced wrist T-score estimates with significant correlation to the DXA-measured values (r = 0.79). Furthermore, by accounting for phalangeal RS parameters, body mass index, and age, a multivariate regression significantly predicted distal radius strength measured in a simulated-fall biomechanical test (r = 0.81). These findings demonstrate the feasibility of interrogating the phalanges using RS for bone quality assessment of distant clinical sites of fragility fractures, such as the wrist. Future work will address transcutaneous measurement challenges as another requirement for scale-up and translation.
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Falanges dos Dedos da Mão , Fraturas por Osteoporose , Humanos , Rádio (Anatomia) , Absorciometria de Fóton/métodos , Antebraço , Cadáver , Densidade ÓsseaRESUMO
Significance: Organelle sizes, which are indicative of cellular status, have implications for drug development and immunology research. At the single cell level, such information could be used to study the heterogeneity of cell response to drugs or pathogens. Aim: Angularly resolved elastic light scattering is known to be sensitive to changes in organelle size distribution. We developed a Mie theory-based simulation of angular scattering from single cells to quantify the effects of noise on scattering and size estimates. Approach: We simulated randomly sampled organelle sizes (drawn from a log normal distribution), interference between different organelles' scattering, and detector noise. We quantified each noise source's effect upon the estimated mean and standard deviation of organelle size distributions. Results: The results demonstrate that signal-to-noise ratio in the angular scattering increased with the number of scatterers, cell area, and exposure time and decreased with the size distribution width. The error in estimating the mean of the size distributions remained below 5% for nearly all experimental parameters tested, but the widest size distribution tested (standard deviation of 600 nm) reached 20%. Conclusions: The simulator revealed that sparse sampling of a broad size distribution can dominate the mismatch between actual and predicted size parameters. Alternative estimation strategies could reduce the discrepancy.
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Luz , Organelas , Simulação por Computador , Razão Sinal-Ruído , Espalhamento de RadiaçãoRESUMO
Angularly-resolved light scattering has been proven to be an early detector of subtle changes in organelle size due to its sensitivity to scatterer size and refractive index contrast. However, for cells immersed in media with a refractive index close to 1.33, the cell itself acts as a larger scatterer and contributes its own angular signature. This whole-cell scattering, highly dependent on the cell's shape and size, is challenging to distinguish from the desired organelle scattering signal. This degrades the accuracy with which organelle size information can be extracted from the angular scattering. To mitigate this effect, we manipulate the refractive index of the immersion medium by mixing it with a water-soluble, biocompatible, high-refractive-index liquid. This approach physically reduces the amount of whole-cell scattering by minimizing the refractive index contrast between the cytosol and the modified medium. We demonstrate this technique on live cells adherent on a coverslip, using Fourier transform light scattering to compute the angular scattering from complex field images. We show that scattering from the cell: media refractive index contrast contributes significant scattering at angles up to twenty degrees and that refractive index-matching reduces such low-angle scatter by factors of up to 4.5. This result indicates the potential of refractive index-matching for improving the estimates of organelle size distributions in single cells.
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Back-illuminated charged-coupled device (BI-CCD) arrays increase quantum efficiency but also amplify etaloning, a multiplicative, wavelength-dependent fixed-pattern effect. When spectral data from hundreds of BI-CCD rows are combined, the averaged spectrum will generally appear etalon-free. This can mask substantial etaloning at the row level, even if the BI-CCD has been treated to suppress the effect. This paper compares two methods of etalon correction, one with simple averaging and one with row-by-row calibration using a fluorescence standard. Two BI-CCD arrays, both roughened by the supplier to reduce etaloning, were used to acquire Raman spectra of murine bone specimens. For one array, etaloning was the dominant source of noise under the exposure conditions chosen, even for the averaged spectrum across all rows; near-infrared-excited Raman peaks were noticeably affected. In this case, row-by-row calibration improved the spectral quality of the average spectrum. The other CCD's performance was shot-noise limited and therefore received no benefit from the extra calibration. The different results highlight the importance of checking for and correcting row-level fixed pattern when measuring weak Raman signals in the presence of a large fluorescence background.
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Artefatos , Análise Espectral Raman , Animais , Calibragem , CamundongosRESUMO
Bone fragility and fracture risk are assessed by measuring the areal bone mineral density (aBMD) using dual-energy X-ray absorptiometry (DXA). While aBMD correlates with bone strength, it is a poor predictor of fragility fracture risk. Alternatively, fracture toughness assesses the bone's resistance to crack propagation and fracture, making it a suitable bone quality metric. Here, we explored how femoral midshaft measurements from DXA, micro-computed tomography (µCT), and Raman spectroscopy could predict fracture toughness. We hypothesized that ovariectomy (OVX) decreases aBMD and fracture toughness compared to controls and we can optimize a multivariate assessment of bone quality by combining results from X-ray and Raman spectroscopy. Female mice underwent an OVX (n = 5) or sham (n = 5) surgery at 3 months of age. Femurs were excised 3 months after ovariectomy and assessed with Raman spectroscopy, µCT, and DXA. Subsequently, a notch was created on the anterior side of the mid-diaphysis of the femurs. Three-point bending induced a controlled fracture that initiated at the notch. The OVX mice had a significantly lower aBMD, cortical thickness, and fracture toughness when compared to controls (p < 0.05). A leave one out cross-validated (LOOCV) partial least squares regression (PLSR) model based only on the combination of aBMD and cortical thickness showed no significant predictive correlations with fracture toughness, whereas a PLSR model based on principal components derived from the full Raman spectra yielded significant prediction (r2 = 0.71, p < 0.05). Further, the PLSR model was improved by incorporating aBMD, cortical thickness, and principal components from Raman spectra (r2 = 0.92, p < 0.001). This exploratory study demonstrates combining X-ray with Raman spectroscopy leads to a more accurate assessment of bone fracture toughness and could be a useful diagnostic tool for the assessment of fragility fracture risk.
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Fraturas do Fêmur , Análise Espectral Raman , Absorciometria de Fóton , Animais , Densidade Óssea , Feminino , Fraturas do Fêmur/diagnóstico por imagem , Humanos , Camundongos , Microtomografia por Raio-XRESUMO
Spatially offset Raman spectroscopy (SORS) is able to detect bone signal transcutaneously and could assist in predicting bone fracture risk. Criteria for optimal source-detector offsets for transcutaneous human measurements, however, are not well-established. Although larger offsets yield a higher percentage of bone signal, the absolute amount of bone signal decreases. Spectral unmixing into bone, adipose, and non-adipose components was employed to quantify changes in bone signal to noise ratio across a range of offsets, and optimal offsets for phalanx and metacarpal measurements were determined. The bone signal to noise ratio was maximized at offsets ranging from 4-6 mm.
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We report what is to our knowledge the first use of Fourier phase microscopy (FPM) to estimate diameters of individual single-micrometer beads and to classify cells based upon changes in scatterer size distribution. FPM, a quantitative phase imaging (QPI) method, combines the planar illumination typically used in off-axis QPI (ideal for Mie theory analysis) with the common-path geometry typically used in on-axis QPI (ideal for optimizing angular scattering range). Low-spatial-frequency imaging artifacts inherent to FPM have negligible impact upon these angular-domain applications. The system is simple to align and stable, and requires no external reference beam. Angular scattering patterns obtained from single 1 µm polystyrene beads in glycerol (Δn=0.11) display unprecedented fidelity to Mie theory, produce diameter estimates consistent with the manufacturer's specifications, and offer precision on the scale of tens of nanometers. Measurements of macrophages at different stages of antibody-dependent cellular phagocytosis demonstrate the ability to detect changes in a cell's scattering caused by the presence of phagocytosed material within.
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Macrófagos/citologia , Microscopia de Contraste de Fase/instrumentação , Espalhamento de Radiação , Animais , Células Cultivadas , Desenho de Equipamento , Análise de Fourier , Luz , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase/métodos , Fagocitose/fisiologia , Poliestirenos , Timócitos/metabolismoRESUMO
Transcutaneous determination of a bone's Raman spectrum is challenging because the type I collagen in the overlying soft tissue is spectroscopically identical to that in bone. In a previous transcutaneous study of murine tibiae, we developed a library-based model called SOLD to unmix spatially offset Raman measurements into three spectra: a bone estimate, a soft tissue estimate, and a residual. Here, we demonstrate the value of combining the bone estimate and the residual to produce a "top layer subtracted" (tls) spectrum. We report superior prediction of two standard bone metrics (volumetric bone mineralization density and maximum torque) using partial least squares regression models based upon tls spectra rather than SOLD bone estimates, implying that the spectral residuals contain useful information. Simulations reinforce experimental in vivo findings. This chemometric approach, which we denote as SOLD/TLS, could have broad applicability in situations where comprehensive spectral libraries are difficult to acquire.
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Osso e Ossos , Análise Espectral Raman , Animais , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Análise dos Mínimos Quadrados , Camundongos , Tíbia/diagnóstico por imagemRESUMO
To better understand the origin of microplastics in municipal drinking water, we evaluated 50 mL water samples from different stages of the City of Rochester's drinking water production and transport route, from Hemlock Lake to the University of Rochester. We directly filtered samples using silicon nitride nanomembrane filters with precisely patterned slit-shaped pores, capturing many of the smallest particulates (<20 µm) that could be absorbed by the human body. We employed machine learning algorithms to quantify the shapes and quantity of debris at different stages of the water transport process, while automatically segregating out fibrous structures from particulate. Particulate concentrations ranged from 13 to 720 particles/mL at different stages of the water transport process and fibrous pollution ranged from 0.4 to 8.3 fibers/mL. A subset of the debris (0.2-8.6%) stained positively with Nile red dye which identifies them as hydrophobic polymers. Further spectroscopic analysis also indicated the presence of many non-plastic particulates, including rust, silicates, and calcium scale. While water leaving the Hemlock Lake facility is mostly devoid of debris, transport through many miles of piping results in the entrainment of a significant amount of debris, including plastics, although in-route reservoirs and end-stage filtration serve to reduce these concentrations.
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Angularly resolved elastic light scattering is an established technique for probing the average size of organelles in biological tissue and cellular ensembles. Focusing of the incident light to illuminate no more than one cell at a time restricts the minimum forward-scattering angle θmin that can be detected. Series of simulated single-cell angular-scattering patterns have been generated to explore how size estimates vary as a function of θmin. At a setting of θmin = 20 deg, the size estimates hop unstably between multiple minima in the solution space as simulated noise (mimicking experimentally observed levels) is varied. As θmin is reduced from 20 deg to 10 deg, the instability vanishes, and the variance of estimates near the correct answer also decreases. The simulations thus suggest that robust Mie theory fits to single-cell scattering at 785 nm excitation require measurements down to at least 15 deg. Notably, no such instability was observed at θmin = 20 deg for narrow bead distributions. Accurate sizing of traditional calibration beads is, therefore, insufficient proof that an angular-scattering system is capable of robust analysis of single cells. Experimental support for the simulation results is also presented using measurements on cells fixed with formaldehyde.
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Elasticidade , Microscopia/métodos , Tamanho das Organelas , Organelas , Animais , Calibragem , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Simulação por Computador , Análise de Fourier , Luz , Camundongos , Mitocôndrias , Modelos Biológicos , Tamanho da Partícula , Poliestirenos/química , Reprodutibilidade dos Testes , Espalhamento de Radiação , Razão Sinal-RuídoRESUMO
Bone strength is a worldwide health concern. Although multiple techniques have been developed to evaluate bone quality, there are still gaps to be filled. Here we report a non-invasive approach for the prediction of bone strength in vivo using spatially offset Raman spectroscopy. Raman spectra were acquired transcutaneously from the tibiae of mice from 4 to 23 weeks old and subsequently on the exposed bones. Partial least squares regression was applied to generate predictions of the areal bone mineral density (aBMD), volumetric bone mineralization density (vBMD), and maximum torque (MT) of each tibia as quantified by dual-energy X-ray absorptiometry, microCT imaging, and biomechanical tests, respectively. Significant correlations were observed between Raman spectral predictions and the reference values in all three categories. To our knowledge, this is the first demonstration of Raman spectroscopy predicting a biomechanical bone parameter (MT) in vivo with an uncertainty much smaller than the spread in the reference values.
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The development of spatially offset Raman spectroscopy (SORS) has enabled deep, non-invasive chemical characterization of turbid media. Here, we use SORS to measure subcortical bone tissue and depth-resolved biochemical variability in intact, exposed murine bones. We also apply the technique to study a mouse model of the genetic bone disorder osteogenesis imperfecta. The results suggest that SORS is more sensitive to disease-related biochemical differences in subcortical trabecular bone and marrow than conventional Raman measurements.
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Osso e Ossos/diagnóstico por imagem , Análise Espectral Raman , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese Imperfeita/diagnóstico por imagem , CoelhosRESUMO
Functional near-infrared spectroscopy (fNIRS) research to date has tended to publish group-averaged rather than individual infant data due to normative basic research goals. Acquisition of individual infant time courses holds interest, however, both for cognitive science and particularly for clinical applications. Infants are more difficult to study than adults as they cannot be instructed to remain still. In addressing this, upright infants pose several associated complications for the researcher. We identified and optimized the factors that affect the quality of fNIRS data from individual 6- to 9-month-old infants exposed to a visual stimulation paradigm. The fNIRS headpiece was reconfigured to reduce inertia, increase comfort, and improve conformity to the head, while preserving fiber density to avoid missing the visual cortex activation. The visual-stimulation protocol was modified to keep the attention of infants throughout the measurement, thus helping to reduce motion artifacts. Adequate optical contact was verified by checking power levels before each measurement. By revising our experimental process and our data rejection criteria to prioritize good optical contact, we report for the first time usable hemodynamic data from 83% of infants and that two-thirds of infants produced a statistically significant fNIRS response.
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Functional near-infrared spectroscopy (fNIRS) records hemodynamic changes in the cortex arising from neurovascular coupling. However, (noninvasive) fNIRS recordings also record surface vascular signals arising from noncortical sources (e.g., in the skull, skin, dura, and other tissues located between the sensors and the brain). A current and important focus in the fNIRS community is determining how to remove these noncortical vascular signals to reduce noise and to prevent researchers from erroneously attributing responses to cortical sources. The current study is the first to test a popular method for removing signals from the surface vasculature (removing short, 1 cm, channel recordings from long, 3 cm, channel recordings) in human infants, a population frequently studied using fNIRS. We find evidence that this method does remove surface vasculature signals and indicates the presence of both local and global surface vasculature signals. However, we do not find that the removal of this information changes the statistical inferences drawn from the data. This latter result not only questions the importance of removing surface vasculature responses for empiricists employing this method, but also calls for future research using other tasks (e.g., ones with a weaker initial result) with this population and possibly additional methods for removing signals arising from the surface vasculature in infants.
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Spintronics use the electron spin as a state variable for information processing and storage. This requires manipulation of spin ensembles for data encoding, and spin transport for information transfer. Because of the central importance of lifetime for understanding and controlling spins, mechanisms that determine this lifetime in bulk systems have been extensively studied. However, a clear understanding of few-spin systems remains challenging. Here, we report spatially resolved magnetic resonance studies of electron spin ensembles confined to a 'spin nanowire' formed by nitrogen ion implantation in diamond. We measure the spin lifetime of the ensemble--that is, its spin autocorrelation time--by monitoring the statistical fluctuations of its net moment, which is in thermal equilibrium and has no imposed polarization gradient. We find that the lifetime of the ensemble is dominated by spin transport from the ensemble into the adjacent spin reservoir that is provided by the remainder of the nanowire. This is in striking contrast to conventional spin-lattice relaxation measurements of isolated spin ensembles. Electron spin resonance spectroscopy performed on nanoscale spin ensembles by means of a novel spin manipulation protocol corroborates spin transport in strong field gradients. Our experiments, supported by microscopic Monte Carlo modelling, provide a unique insight into the intrinsic dynamics of pure spin currents needed for nanoscale devices that seek to control spins.
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In functional near-infrared spectroscopy (fNIRS) of human cerebral hemodynamics, dedicated surface-sensitive recording channels are useful for regressing out background hemodynamics and isolating activation-specific responses. A wide variety of source-detector separations have been utilized for this purpose. Here, we report a direct comparison of regression performance between two extremes of the reported range, 13 and 6 mm. Measurements of visual stimulation response (flickering radial checkerboard) were obtained from nine adults using a standard commercial source-detector grid with 13-mm diagonals, into which three extra detector fibers were placed to provide 6-mm channels at certain locations. When the NIRS recordings (17 total trials) were processed, the contrast-to-noise ratio was significantly higher with 6-mm regression channels than with 13 mm. The advantage could be due in part to the undesired sensing of brain activity by the 13-mm channels. We suggest that shorter distances be considered for optimal removal of superficial hemodynamics in NIRS signals from the adult brain.
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Understanding the complex properties of electronic and spintronic devices at the micro- and nano-scale is a topic of intense current interest as it becomes increasingly important for scientific progress and technological applications. In operando characterization of such devices by scanning probe techniques is particularly well-suited for the microscopic study of these properties. We have developed a scanning probe microscope (SPM) which is capable of both standard force imaging (atomic, magnetic, electrostatic) and simultaneous electrical transport measurements. We utilize flexible and inexpensive FPGA (field-programmable gate array) hardware and a custom software framework developed in National Instrument's LabVIEW environment to perform the various aspects of microscope operation and device measurement. The FPGA-based approach enables sensitive, real-time cantilever frequency-shift detection. Using this system, we demonstrate electrostatic force microscopy of an electrically biased graphene field-effect transistor device. The combination of SPM and electrical transport also enables imaging of the transport response to a localized perturbation provided by the scanned cantilever tip. Facilitated by the broad presence of LabVIEW in the experimental sciences and the openness of our software solution, our system permits a wide variety of combined scanning and transport measurements by providing standardized interfaces and flexible access to all aspects of a measurement (input and output signals, and processed data). Our system also enables precise control of timing (synchronization of scanning and transport operations) and implementation of sophisticated feedback protocols, and thus should be broadly interesting and useful to practitioners in the field.
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We present an angular-scattering optical method that is capable of measuring the mean size of scatterers in static ensembles within a field of view less than 20 µm in diameter. Using interferometry, the method overcomes the inability of intensity-based models to tolerate the large speckle grains associated with such small illumination areas. By first estimating each scatterer's location, the method can model between-scatterer interference as well as traditional single-particle Mie scattering. Direct angular-domain measurements provide finer angular resolution than digitally transformed image-plane recordings. This increases sensitivity to size-dependent scattering features, enabling more robust size estimates. The sensitivity of these angular-scattering measurements to various sizes of polystyrene beads is demonstrated. Interferometry also allows recovery of the full complex scattered field, including a size-dependent phase profile in the angular-scattering pattern.
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Interferometria/instrumentação , Lentes , Refratometria/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Espalhamento de RadiaçãoRESUMO
Clinical diagnoses of bone health and fracture risk typically rely on measurements of bone density or structure, but the strength of a bone is also dependent on its chemical composition. Raman spectroscopy has been used extensively in ex vivo studies to measure the chemical composition of bone. Recently, spatially offset Raman spectroscopy (SORS) has been utilized to measure bone transcutaneously. Although the results are promising, further advancements are necessary to make noninvasive, in vivo measurements of bone with SORS that are of sufficient quality to generate accurate predictions of fracture risk. In order to separate the signals from bone and soft tissue that contribute to a transcutaneous measurement, we developed an overconstrained extraction algorithm that is based on fitting with spectral libraries. This approach allows for accurate spectral unmixing despite the fact that similar chemical components (e.g., type I collagen) are present in both bone and soft tissue. The algorithm was utilized to transcutaneously detect biochemical differences in the tibiae of wild-type mice between 1 and 7 months of age and between the tibiae of wild-type mice and a mouse model of osteogenesis imperfecta. These results represent the first diagnostically sensitive, transcutaneous measurements of bone using SORS.