Line-scan Imaging Research Articles

Oxidation protection of Ti–6Al–4V alloy using a novel glass–amorphous silica composite coating

A novel glass–amorphous silica composite coating was prepared by the slurry method in order to improve the oxidation resistance of Ti–6Al–4V alloy at high temperatures. The microstructure of the as-prepared composite coating was analyzed by SEM, XRD and EDS techniques. The oxidation resistance and the microstructure evolution of the composite coating at 800°C for 50h were also studied. The results show that mass gains of the specimens coated with the composite coating were far less than that of the uncoated ones after oxidation of 50h. Thick oxide scales composed of plate-like rutile TiO2 and some granular α-Al2O3 formed on bare Ti–6Al–4V alloy, while quartz, cristobalite and diopside were observed in the composite coating, which are useful crystals for slowing the inward diffusion of oxygen to the substrate. The cross-sectional EDS line scanning images show that inward diffusion of oxygen and outward diffusion of Ti through the composite coating were insignificant. The microhardness profile reveals that the solid solution oxygen in Ti–6Al–4V alloy with composite coating was limited.

Real-time 1D hyperspectral imaging of porous silicon-based photonic crystals with one-dimensional chemical composition gradients undergoing pore-filling-induced spectral shifts

A line-scan hyperspectral imager can provide real-time spectral information at many points along a transect of a gradient-modified material. This is demonstrated by monitoring the optical reflectance spectrum of a porous silicon photonic crystal with a hydrophobic/hydrophilic pore-wall surface composition gradient while it is immersed in water to which ethanol is added. The combination of the hyperspectral imager and the pore-wall composition gradient allows a detailed study of the pore filling process. It is observed that even the most hydrophobic pores fill to a small extent when only a small amount of ethanol has been added. This gradient of filling is in contrast to visible observation of the porous silicon samples, which can suggest a step change in pore filling. The pore-filling process can take many minutes when the solution composition allows pores at a given position along the gradient to be about 50% filled. These observations demonstrate the utility of aligning a line-scan hyperspectral imager with a gradient material.

Quantitative Determination of Onion Internal Quality Using Reflectance, Interactance, and Transmittance Modes of Hyperspectral Imaging

<abstract><title><italic>Abstract.</italic></title> The internal quality of onions is important to both consumers and onion processors, but current methods for evaluating the internal quality are mostly destructive. The overall goal of this study was to investigate the feasibility of using hyperspectral imaging technology to quantitatively predict the amount of dry matter, the soluble solids content, and the firmness of onions. A total of 308 onions were scanned using a line-scan hyperspectral imaging system with three sensing modes (reflectance, interactance, and transmittance) in the spectral region of 400-1000 nm. An ellipsoidal model was developed to correct the uneven illumination caused by the curvature of the surface in the reflectance images. The spectra extracted from onion spectral images were used to develop partial least squares (PLS) regression models. Results showed that interactance achieved comparable or even better results than transmittance, and the two modes performed significantly better than diffuse reflectance. In interactance mode, soluble solids content [coefficient of determination (R²) = 0.93, standard error of prediction (SEP) = 1.46 °Brix] and dry matter (R² = 0.93, SEP = 1.61%) can be estimated better than firmness (R² = 0.59, SEP = 9.75 N). This study demonstrated for the first time that the interactance mode of the hyperspectral imaging technique can be used to quantitatively predict the internal quality properties of an onion. The lab-based hyperspectral imaging system has the potential to be used in an automated online quality inspection system for predicting the internal quality of onions.

Development of Multispectral Algorithm for Detection of Cracked Tomatoes

<abstract><title><italic>Abstract.</italic></title> <bold> </bold>Tomato cracks present a potential food safety risk by allowing internalization of pathogens. This article reports a first-step study for the development and evaluation of a simple multispectral algorithm for the detection of defect cracked tomatoes. Analysis of hyperspectral visible/near-infrared tomato images identified two wavebands, 749 nm and 887 nm, for computation of two decision functions to detect defect cracked tomatoes and to differentiate them from normal tomatoes. Detection of defect cracked tomatoes was based on spectral identification of non-skin pixels coupled with quantitative evaluation of the number of non-skin pixels compared to the total image pixels for each tomato. The algorithm successfully classified 94% of the normal tomatoes and 99% of the defect tomatoes used in this study. The results suggest that this multispectral spectral imaging-based method has potential for implementation in line-scan imaging systems as a tool for rapid food inspection on high-speed tomato processing lines.

A Method of Combining Laser Data and Line Scanning Images

A Method of Combining Laser Data and Line Scanning Images

Stripe Noise Removal from Line Scan Images of Foreign Fibers

Stripe Noise Removal from Line Scan Images of Foreign Fibers

Quantitative analysis of calcium spikes in noisy fluorescent background.

Intracellular calcium signals are studied by laser-scanning confocal fluorescence microscopy. The required spatio-temporal resolution makes description of calcium signals difficult because of the low signal-to-noise ratio. We designed a new procedure of calcium spike analysis based on their fitting with a model. The accuracy and precision of calcium spike description were tested on synthetic datasets generated either with randomly varied spike parameters and Gaussian noise of constant amplitude, or with constant spike parameters and Gaussian noise of various amplitudes. Statistical analysis was used to evaluate the performance of spike fitting algorithms. The procedure was optimized for reliable estimation of calcium spike parameters and for dismissal of false events. A new algorithm was introduced that corrects the acquisition time of pixels in line-scan images that is in error due to sequential acquisition of individual pixels along the space coordinate. New software was developed in Matlab and provided for general use. It allows interactive dissection of temporal profiles of calcium spikes from x-t images, their fitting with predefined function(s) and acceptance of results on statistical grounds, thus allowing efficient analysis and reliable description of calcium signaling in cardiac myocytes down to the in situ function of ryanodine receptors.

Synchrony of Cardiomyocyte Ca2+ Release is Controlled by t-tubule Organization, SR Ca2+ Content, and Ryanodine Receptor Ca2+ Sensitivity

Recent work has demonstrated that cardiomyocyte Ca2+release is desynchronized in several pathological conditions. Loss of Ca2+ release synchrony has been attributed to t-tubule disruption, but it is unknown if other factors also contribute. We investigated this issue in normal and failing myocytes by integrating experimental data with a mathematical model describing spatiotemporal dynamics of Ca2+ in the cytosol and sarcoplasmic reticulum (SR). Heart failure development in postinfarction mice was associated with progressive t-tubule disorganization, as quantified by fast-Fourier transforms. Data from fast-Fourier transforms were then incorporated in the model as a dyadic organization index, reflecting the proportion of ryanodine receptors located in dyads. With decreasing dyadic-organization index, the model predicted greater dyssynchrony of Ca2+ release, which exceeded that observed in experimental line-scan images. Model and experiment were reconciled by reducing the threshold for Ca2+ release in the model, suggesting that increased RyR sensitivity partially offsets the desynchronizing effects of t-tubule disruption in heart failure. Reducing the magnitude of SR Ca2+ content and release, whether experimentally by thapsigargin treatment, or in the model, desynchronized the Ca2+ transient. However, in cardiomyocytes isolated from SERCA2 knockout mice, RyR sensitization offset such effects. A similar interplay between RyR sensitivity and SR content was observed during treatment of myocytes with low-dose caffeine. Initial synchronization of Ca2+ release during caffeine was reversed as SR content declined due to enhanced RyR leak. Thus, synchrony of cardiomyocyte Ca2+ release is not only determined by t-tubule organization but also by the interplay between RyR sensitivity and SR Ca2+ content.

Near-infrared hyperspectral imaging in tandem with partial least squares regression and genetic algorithm for non-destructive determination and visualization of Pseudomonas loads in chicken fillets

Hyperspectral imaging was exploited for its potential in direct and fast determination of Pseudomonas loads in raw chicken breast fillets. A line-scan hyperspectral imaging system (900–1700nm) was employed to obtain sample images, which were then further corrected, modified and processed. The prepared images were correlated with the true Pseudomonas counts of these samples using partial least squares (PLS) regression. To enhance model performance, different spectral extraction approaches, spectral preprocessing methods as well as wavelength selection schemes based on genetic algorithm were investigated. The results revealed that extraction of mean spectra is more efficient for representation of sample spectra than computation of median spectra. The best full wavelength model was attained based on spectral images preprocessed with standard normal variate, and the correlation coefficients (R) and root mean squared errors (RMSEs) for the model were above 0.81 and below 0.80log10CFUg−1, respectively. In development of simplified models, wavelengths were selected by using a proposed two-step method based on genetic algorithm. The best model utilized only 14 bands in five segments and produced R and RMSEs of 0.91 and 0.55log10CFUg−1, 0.87 and 0.65log10CFUg−1 as well as 0.88 and 0.64log10CFUg−1 for calibration, cross-validation and prediction, respectively. Moreover, the prediction maps offered a novel way for visualizing the gradient of Pseudomonas loads on meat surface. Hyperspectral imaging is demonstrated to be an effective tool for nondestructive measurement of Pseudomonas in raw chicken breast fillets.

Cell Edge Detector: Recovering Length Information from Line-Scan Confocal Images

Confocal microscopy has greatly enhanced our understanding of cardiac excitation-contraction coupling. Linescan images have been used to obtain detailed information about intracellular calcium concentration disregarding the mechanical activity of the cell. In order to fill this gap we have designed an algorithm to recover the cell length information from those images. We implemented this program using LabVIEW language. An intuitive user interface allows the researcher to load the image; scan the longitudinal axis; select portion of the file; detect the borders and plot the calcium and cell shortening traces with the corresponding shortening versus calcium loop. The software also calculates and plots into a table the following parameters : maximum fluorescence (F/F0), decay time (tau), peak fractional shortening (L%),half time to relaxation (t1/2), maximum dL/dt, and from the loop curve the calcium concentration corresponding to half shortening (EC50). This new approach allows us to obtain richer information from the linescan images by extracting the corresponding shortening traces from the fluorescence signal. This software is already operational in our labs and has been used to analyze confocal images of calcium transients and myocyte contraction in cardiac cells.

Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm

Neural activity leads to hemodynamic changes which can be detected by functional magnetic resonance imaging (fMRI). The determination of blood flow changes in individual vessels is an important aspect of understanding these hemodynamic signals. Blood flow can be calculated from the measurements of vessel diameter and blood velocity. When using line-scan imaging, the movement of blood in the vessel leads to streaks in space-time images, where streak angle is a function of the blood velocity. A variety of methods have been proposed to determine blood velocity from such space-time image sequences. Of these, the Radon transform is relatively easy to implement and has fast data processing. However, the precision of the velocity measurements is dependent on the number of Radon transforms performed, which creates a trade-off between the processing speed and measurement precision. In addition, factors like image contrast, imaging depth, image acquisition speed, and movement artifacts especially in large mammals, can potentially lead to data acquisition that results in erroneous velocity measurements. Here we show that pre-processing the data with a Sobel filter and iterative application of Radon transforms address these issues and provide more accurate blood velocity measurements. Improved signal quality of the image as a result of Sobel filtering increases the accuracy and the iterative Radon transform offers both increased precision and an order of magnitude faster implementation of velocity measurements. This algorithm does not use a priori knowledge of angle information and therefore is sensitive to sudden changes in blood flow. It can be applied on any set of space-time images with red blood cell (RBC) streaks, commonly acquired through line-scan imaging or reconstructed from full-frame, time-lapse images of the vasculature.

The Structure-Function Relationship between Macular Morphology and Visual Function Analyzed by Optical Coherence Tomography in Retinitis Pigmentosa

Purpose. To evaluate the relationship between macular microstructures and visual function in retinitis pigmentosa (RP). Method. Fourier domain optical coherence tomography (FD-OCT) and Goldmann perimetry were used to examine 100 eyes of 100 RP patients. The preserved photoreceptor outer segment (PROS) length was measured at the horizontal and vertical high definition line scans. The PROS area was calculated from slab image and line scans simultaneously. The visual field area (VFA) was quantified. Each retinal thickness was measured: inner retina (IRT), outer retina (ORT), subfoveal choroidal thickness (SFCT), and central retinal thickness (CRT). Results. The PROS area values acquired differently were consistent. The VFA was related significantly to the CRT, ORT, PROS length (vertical and horizontal), and PROS area (line scan and slab image). Visual acuity was correlated with the CRT, ORT, IRT, PROS length (horizontal and vertical), and PROS area (line scan and slab image) significantly. Multiple linear regression analysis revealed that the PROS horizontal length and ORT were related to the VFA and visual acuity, respectively. Conclusion. Among the macular microstructures, the PROS horizontal length and the ORT were most correlated with VFA and visual acuity, respectively. However, SFCT is not related to visual function.

Efficient Automatic Analysis of High-Speed Confocal Images Containing Localized Calcium Release Events

High-speed confocal microscopy is a relatively new method used to investigate localized calcium release events, most notably calcium sparks. Line scan images can reliably be recorded at speeds up to 60000 lines/s while for X-Y images 120 frame/s rate is achievable. The increased amount of recorded data raises the necessity of efficient automatic analysis methods. A set of images recorded on frog skeletal muscle cells after caffeine treatment was used to test the described methods.We present a collection of methods useful for the automatic analysis of high speed line scan and X-Y recordings, mostly based on stationary wavelet transform (SWT). Our one-dimensional SWT based ember-detection method was adapted to detect sparks on high-speed line scan recordings. The centerline of detected sparks is automatically marked on each scan line to assess the spatial and temporal characteristics of the spark. 3.66% of all sparks (n=23704) in the test image set had complex spatial properties revealed by high-speed imaging. Amplitude and full width at half maximum (FWHM) is determined for each scan line to calculate the amount of calcium released (signal mass).On X-Y image series two-dimensional SWT was used to denoise the images and to detect sparks on each frame. The orientation of the skeletal muscle cell and the position of sarcomeres are determined using fast Fourier-transform on the stationary components of the image series. The position of the center for each spark is determined relatively to the sarcomeres. FWHM is calculated in the axes perpendicular (FWHMX) and parallel (FWHMY) to the sarcomeres. For the test dataset (n=22426), FWHMX/FWHMY is 1.22.The above-described methods enable us to automatically analyze high-speed confocal images and to reveal some of their properties which were hidden on conventional confocal images.

Epidermal keratinocyte polarity and motility require Ca2+ influx through TRPV1

Ca(2+) has long been known to play an important role in cellular polarity and guidance. We studied the role of Ca(2+) signaling during random and directed cell migration to better understand whether Ca(2+) directs cell motility from the leading edge and which ion channels are involved in this function by using primary zebrafish keratinocytes. Rapid line-scan and time-lapse imaging of intracellular Ca(2+) (Ca(2+)i) during migration and automated image alignment enabled us to characterize and map the spatiotemporal changes in Ca(2+)i. We show that asymmetric distributions of lamellipodial Ca(2+) sparks are encoded in frequency, not amplitude, and that they correlate with cellular rotation during migration. Directed migration during galvanotaxis increases the frequency of Ca(2+) sparks over the entire lamellipod; however, these events do not give rise to asymmetric Ca(2+)i signals that correlate with turning. We demonstrate that Ca(2+)-permeable channels within these cells are mechanically activated and include several transient receptor potential family members, including TRPV1. Last, we demonstrate that cell motility and Ca(2+)i activity are affected by pharmacological agents that target TRPV1, indicating a novel role for this channel during cell migration.

Mapping of egg yolk and animal skin glue paint binders in Early Renaissance paintings using near infrared reflectance imaging spectroscopy

In situ chemical imaging techniques are being developed to provide information on the spatial distribution of artists' pigments used in polychrome works of art such as paintings. The new methods include reflectance imaging spectroscopy and X-ray fluorescence mapping. Results from these new methods have extended the knowledge obtained from site-specific chemical analyses widely in use. While these mapping methods have aided in determining the distribution of pigments, there is a growing interest to develop methods capable of identifying and mapping organic paint binders as well. Near infrared (NIR) reflectance spectroscopy has been extensively used in the remote sensing field as well as in the chemical industry to detect organic compounds. NIR spectroscopy provides a rapid method to assay organics by utilizing vibrational overtones and combination bands of fundamental absorptions that occur in the mid-IR. Here we explore the utility of NIR reflectance imaging spectroscopy to map organic binders in situ by examining a series of panel paintings known to have been painted using distemper (animal skin glue) and tempera (egg yolk) binders as determined by amino acid analysis of samples taken from multiple sites on the panels. In this report we demonstrate the success in identifying and mapping these binders by NIR reflectance imaging spectroscopy in situ. Three of the four panel paintings from Cosimo Tura's The Annunciation with Saint Francis and Saint Louis of Toulouse (ca. 1475) are imaged using a highly sensitive, line-scanning hyperspectral imaging camera. The results show an animal skin glue binder was used for the blue skies and blue robe of the Virgin Mary, and egg yolk tempera was used for the red robes and brown landscape. The mapping results show evidence for the use of both egg yolk and animal skin glue in the faces of the figures. The strongest absorption associated with lipidic egg yolk features visually correlates with areas that appear to have white highlights. The results are in agreement with prior site-specific amino acid analysis, underscoring the synergy of both methods. The work here demonstrates that NIR reflectance imaging spectroscopy is a useful technique that can identify and map paint binding media based on differences in chemical composition.

基于线扫描相位差分成像的光学元件激光损伤快速检测技术

基于线扫描相位差分成像的光学元件激光损伤快速检测技术

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Properties of Calcium Transients in Rabbit Atrial Myocytes

In comparison to ventricular myocytes, there is relatively little information regarding the transport mechanisms underlying the Ca2+ transient in atrial cells. In this study, we focus on the contribution of the sarcoplasmic Ca-ATPase (SERCA) and Na/Ca exchanger (NCX) to the removal of Ca2+ from the cytosol in atrial myocytes. Isolated rabbit atrial myocytes were loaded with fluo-4-AM and superfused with a Tyrode's solution at room temperature (20 – 24 °C) in a chamber on a Zeiss Pascal LSM5 confocal microscope. Cells were electrically stimulated at 1 Hz via bath electrodes and linescan images obtained by scanning transversely across the cell. Electrically stimulated Ca2+ transients were initiated at the periphery of the cell, rising to a peak within 66.5 ± 15.4 ms. The peak of the transient at the center of the cell was delayed and had reduced amplitude compared with the transient at the cell edge. The decay phase of the twitch transient averaged across the cell width was fitted by a single exponential (ktwitch = 1.71 ± 0.26 s−1). Rapid application of 10 mM caffeine to unload the sarcoplasmic reticulum (SR) produced a large transient that decayed with a rate constant (kcaff) of 0.19 ± 0.01 s−1. Following the washout of caffeine, twitch Ca2+ transients were markedly diminished but recovered to a steady-state within ∼20 s. Subsequent rapid application of caffeine in the presence of Ni2+ (10 mM) produced a large Ca2+ transient that recovered with a rate constant, kNi, of 0.02 ± 0.004 s−1. SERCA, NCX and slow pathways were calculated to contribute, respectively, 87.4 ± 1.8%, 10.8 ± 1.4% and 1.8± 0.5% of the total Ca2+ flux. In conclusion, the overall calcium extrusion pattern appears similar to ventricular myocytes.

CaMKII-Dependent Phosphorylation Modulates Ca2+ Cycling in Sinoatrial Node Cells to Regulate Cardiac Pacemaker Function

Spontaneous beating of rabbit sinoatrial node cells (SANC) is linked to rhythmic, submembrane, sarcoplasmic reticulum (SR) generated local Ca2+releases (LCR), which activate inward Na+/Ca2+exchange current to impart exponential increase of the late diastolic depolarization rate and fire an action potential (AP). Rabbit SANC have high basal PKA-dependent phosphorylation, but basal CaMKII-dependent phosphorylation has never been ascertained. Here we show that SANC maintain high basal PLB phosphorylation at Thr17 site (western blotting), employed as a marker of CaMKII-dependent protein phosphorylation, that is markedly suppressed by CaMKII inhibitor, KN-93, but not its inactive analog KN-92. Considering that CaMKII activity is critically dependent on intracellular [Ca2+]i we employed BAPTA-AM to chelate [Ca2+]i. Both KN-93 and BAPTA-AM markedly decrease the LCR size (confocal line-scan imaging), LCR number per each spontaneous cycle and prolong the LCR period (time between AP-induced Ca2+ transient and subsequent LCR). The increase in the LCR period predicts concurrent increase in the spontaneous cycle length. To delineate mechanisms of CaMKII-dependent regulation of Ca2+ cycling, avoiding interference of L-type Ca2+channels, which are also regulated by CaMKII, we permeabilized SANC with saponin. Elevation of cytosolic [Ca2+] from 50 to 150 nmol/L in permeabilized SANC significantly increases the LCR size and amplitude due, in part, to a marked increase of PLB phosphorylation at Thr17site and resultant increase in the SR Ca2+ content and, in part, to RyR phosphorylation at PKA- and CaMKII-dependent Ser2809 site (immunostaining). CaMKII-inhibitor AIP suppresses LCR characteristics in permeabilized SANC, decreases phosphorylation of RyR, PLB and reduces the SR Ca2+ load. Thus, normal automaticity of SANC is regulated by basal CaMKII-dependent phosphorylation, partly via modulation of intrinsic SR Ca2+ cycling, i.e. SR Ca2+ pumping and release attained through phosphorylation of PLB and RyR.

Automated Model-Based Calibration of Short-Wavelength Infrared (SWIR) Imaging Spectrographs

Among the variety of available hyperspectral imaging systems, the line-scan technique stands out for its short acquisition time and good signal-to-noise ratio. However, due to imperfections in the camera lens and, in particular, optical components of the imaging spectrograph, the acquired images are spatially and spectrally distorted, which can significantly degrade the accuracy of the subsequent hyperspectral image analysis. In this work, we propose and evaluate an automated method for correction of spatial and spectral distortions introduced by a line-scan hyperspectral imaging system operating in the short wavelength infrared (SWIR) spectral range from 1000 nm to 2500 nm. The proposed method is based on non-rigid registration of the distorted and reference images corresponding to two passive calibration objects. The results of the validation show that the proposed method is accurate, efficient, and applicable for calibration of line-scan hyperspectral imaging systems. Moreover, the design of the method and of the calibration objects allows integration with systems operating in diffuse reflectance or transmittance modes.