Orientation Prior and Consistent Model Selection Increase Sensitivity of Tract-Based Spatial Statistics in Crossing-Fiber Regions.

The goal of this paper is to increase the statistical power of crossing-fiber statistics in voxelwise analyses of diffusion-weighted magnetic resonance imaging (DW-MRI) data. In the proposed framework, a fiber orientation atlas and a model complexity atlas were used to fit the ball-and-sticks model to diffusion-weighted images of subjects in a prospective population-based cohort study. Reproducibility and sensitivity of the partial volume fractions in the ball-and-sticks model were analyzed using TBSS (tract-based spatial statistics) and compared to a reference framework. The reproducibility was investigated on two scans of 30 subjects acquired with an interval of approximately three weeks by studying the intraclass correlation coefficient (ICC). The sensitivity to true biological effects was evaluated by studying the regression with age on 500 subjects from 65 to 90 years old. Compared to the reference framework, the ICC improved significantly when using the proposed framework. Higher t-statistics indicated that regression coefficients with age could be determined more precisely with the proposed framework and more voxels correlated significantly with age. The application of a fiber orientation atlas and a model complexity atlas can significantly improve the reproducibility and sensitivity of crossing-fiber statistics in TBSS.

Comparison between dynamic gadoxetate-enhanced MRI and 99mTc-mebrofenin hepatobiliary scintigraphy with SPECT for quantitative assessment of liver function.

OBJECTIVES: To compare Gd-EOB-DTPA dynamic hepatocyte-specific contrast-enhanced MRI (DHCE-MRI) with 99mTc-mebrofenin hepatobiliary scintigraphy (HBS) as quantitative liver function tests for the preoperative assessment of patients undergoing liver resection. METHODS: Patients undergoing liver surgery and preoperative assessment of future remnant liver (FRL) function using 99mTc-mebrofenin HBS were included. Patients underwent DHCE-MRI. Total liver uptake function was calculated for both modalities: mebrofenin uptake rate (MUR) and Ki respectively. The FRL was delineated with both SPECT-CT and MRI to calculate the functional share. Blood samples were taken to assess biochemical liver parameters. RESULTS: A total of 20 patients were included. The HBS-derived MUR and the DHCE-MRI-derived mean Ki correlated strongly for both total and FRL function (Pearson r = 0.70, p = 0.001 and r = 0.89, p < 0.001 respectively). There was a strong agreement between the functional share determined with both modalities (ICC = 0.944, 95% CI 0.863-0.978, n = 20). There was a significant negative correlation between liver aminotransferases and bilirubin for both MUR and Ki. CONCLUSIONS: Assessment of liver function with DHCE-MRI is comparable with that of 99mTc-mebrofenin HBS and has the potential to be combined with diagnostic MRI imaging. This can therefore provide a one-stop-shop modality for the preoperative assessment of patients undergoing liver surgery. KEY POINTS: • Quantitative assessment of liver function using hepatobiliary scintigraphy is performed in the preoperative assessment of patients undergoing liver surgery in order to prevent posthepatectomy liver failure. • Gd-EOB-DTPA dynamic hepatocyte-specific contrast-enhanced MRI (DHCE-MRI) is an emerging method to quantify liver function and can serve as a potential alternative to hepatobiliary scintigraphy. • Assessment of liver function with dynamic gadoxetate-enhanced MRI is comparable with that of hepatobiliary scintigraphy and has the potential to be combined with diagnostic MRI imaging.

A toolkit for the characterization of CCD cameras for transmission electron microscopy.

Charge-coupled devices (CCD) are nowadays commonly utilized in transmission electron microscopy (TEM) for applications in life sciences. Direct access to digitized images has revolutionized the use of electron microscopy, sparking developments such as automated collection of tomographic data, focal series, random conical tilt pairs and ultralarge single-particle data sets. Nevertheless, for ultrahigh-resolution work photographic plates are often still preferred. In the ideal case, the quality of the recorded image of a vitrified biological sample would solely be determined by the counting statistics of the limited electron dose the sample can withstand before beam-induced alterations dominate. Unfortunately, the image is degraded by the non-ideal point-spread function of the detector, as a result of a scintillator coupled by fibre optics to a CCD, and the addition of several inherent noise components. Different detector manufacturers provide different types of figures of merit when advertising the quality of their detector. It is hard for most laboratories to verify whether all of the anticipated specifications are met. In this report, a set of algorithms is presented to characterize on-axis slow-scan large-area CCD-based TEM detectors. These tools have been added to a publicly available image-processing toolbox for MATLAB. Three in-house CCD cameras were carefully characterized, yielding, among others, statistics for hot and bad pixels, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. These statistics will aid data-collection strategy programs and provide prior information for quantitative imaging. The relative performance of the characterized detectors is discussed and a comparison is made with similar detectors that are used in the field of X-ray crystallography.

DNA deformations near charged surfaces: electron and atomic force microscopy views.

DNA is a very important cell structural element, which determines the level of expression of genes by virtue of its interaction with regulatory proteins. We use electron (EM) and atomic force microscopy (AFM) to characterize the flexibility of double-stranded DNA ( approximately 150-950 nm long) close to a charged surface. Automated procedures for the extraction of DNA contours ( approximately 10-120 nm for EM data and approximately 10-300 nm for AFM data) combined with new statistical chain descriptors indicate a uniquely two-dimensional equilibration of the molecules on the substrate surface regardless of the procedure of molecule mounting. However, in contrast to AFM, the EM mounting leads to a noticeable decrease in DNA persistence length together with decreased kurtosis. Analysis of local bending on short length scales (down to 6 nm in the EM study) shows that DNA flexibility behaves as predicted by the wormlike chain model. We therefore argue that adhesion of DNA to a charged surface may lead to additional static bending (kinking) of approximately 5 degrees per dinucleotide step without impairing the dynamic behavior of the DNA backbone. Implications of this finding are discussed.

Quantitative image analysis for evaluating the coating thickness and pore distribution in coated small particles.

PURPOSE: This study aims to develop a characterization method for coating structure based on image analysis, which is particularly promising for the rational design of coated particles in the pharmaceutical industry. METHODS: The method applies the MATLAB image processing toolbox to images of coated particles taken with Confocal Laser Scanning Microscopy (CSLM). The coating thicknesses have been determined along the particle perimeter, from which a statistical analysis could be performed to obtain relevant thickness properties, e.g. the minimum coating thickness and the span of the thickness distribution. The characterization of the pore structure involved a proper segmentation of pores from the coating and a granulometry operation. RESULTS: The presented method facilitates the quantification of porosity, thickness and pore size distribution of a coating. These parameters are considered the important coating properties, which are critical to coating functionality. Additionally, the effect of the coating process variations on coating quality can straight-forwardly be assessed. CONCLUSIONS: Enabling a good characterization of the coating qualities, the presented method can be used as a fast and effective tool to predict coating functionality. This approach also enables the influence of different process conditions on coating properties to be effectively monitored, which latterly leads to process tailoring.

Protrusion method for automated estimation of polyp size on CT colonography.

OBJECTIVE: The purpose of this study was to assess the accuracy and measurement variability of automated lesion measurement on CT colonography in comparison with manual 2D and 3D techniques under varying scanning conditions. MATERIALS AND METHODS: The study included phantoms (23 phantom objects) and patients (16 polyps). Measurement with sliding calipers served as the reference for the phantom data. The mean of two independent colonoscopic measurements was the reference for the polyps. The automated measurement was developed for a computer-aided detection scheme, and the size of any detected object was obtained from measurement of its largest diameter. The automated measurement was compared with manual 2D and 3D measurements by two experienced observers. RESULTS: For phantom data, the measurement variability of the automated method was significantly less than that of the two observers (p < 0.05), except for the 3D measurement by observer 1, as follows: automated, 0.86 mm; observer 1, 1.76 mm (2D), 0.96 (3D); observer 2, 1.34 mm (2D), 1.45 mm (3D). The variability of the automated method did not differ significantly from that of manual methods in measurement with patient data. The automated method had a systematic error for phantom data (1.9 mm). CONCLUSION: For phantoms, the automated method has less measurement variability than manual 2D and 3D techniques. For true polyps, the measurement variability of the automated method is comparable with that of manual methods. The automated method does not suffer from intraobserver variability. Because systematic error can be calibrated, automated size measurement may contribute to a practical evaluation strategy.

Shaving diffusion tensor images in discriminant analysis: a study into schizophrenia.

A technique called 'shaving' is introduced to automatically extract the combination of relevant image regions in a comparative study. No hypothesis is needed, as in conventional pre-defined or expert selected region of interest (ROI)-analysis. In contrast to traditional voxel based analysis (VBA), correlations within the data can be modeled using principal component analysis (PCA) and linear discriminant analysis (LDA). A study into schizophrenia using diffusion tensor imaging (DTI) serves as an application. Conventional VBA found a decreased fractional anisotropy (FA) in a part of the genu of the corpus callosum and an increased FA in larger parts of white matter. The proposed method reproduced the decrease in FA in the corpus callosum and found an increase in the posterior limb of the internal capsule and uncinate fasciculus. A correlation between the decrease in the corpus callosum and the increase in the uncinate fasciculus was demonstrated.

Aerating grassland before manure application reduces runoff nutrient loads in a high rainfall environment.

The effect of mechanically aerating grassland before liquid manure application in the fall on surface runoff and transport of nutrients and solids was studied in a high rainfall area. The two treatments were control and aeration, the latter receiving one pass with an aerator perpendicular to the slope before fall application of liquid manure (dairy in Years 1-3 and swine in Year 4). Treatments were randomly assigned on 3 to 5% sloping land with a silt loam surface soil (Aquic Dystroxerept) planted in orchardgrass (Dactylis glomerata L.). Runoff from natural rainfall events was sampled for nutrient and solids analysis. Aeration significantly reduced runoff and loads of suspended solids, total Kjeldahl N (TKN), and dissolved reactive P in all years. Annual runoff amounts were reduced by 47 to 81%, suspended and volatile solid loads by 48 to 69% and 42 to 83%, respectively, TKN loads by 56 to 81%, and total P (TP) loads by 25 to 75%. Loads of the soluble nutrient NH4-N, dissolved reactive P, and K were reduced by 41 to 83%. The first three runoff events after manure application accounted for approximately one-third of the annual total runoff and solid and nutrient loads when averaged across treatments, with loads of TKN, K, and NH4-N totaling 4.4, 3.3, and 1.9 kg ha-1, respectively. Aeration slightly increased downward movement of NO3-N, but not other nutrients in the soil. Thus mechanical aeration can be an effective tool for reducing runoff and loads of solids and nutrients after surface application of liquid manure on sloping grassland.

Using line segments as structuring elements for sampling-invariant measurements.

When performing measurements in digitized images, the pixel pitch does not necessarily limit the attainable accuracy. Proper sampling of a band-limited continuous-domain image preserves all information present in the image prior to digitization. It is therefore (theoretically) possible to obtain measurements from the digitized image that are identical to measurements made in the continuous domain. Such measurements are sampling invariant, since they are independent of the chosen sampling grid. It is impossible to attain strict sampling invariance for filters in mathematical morphology due to their nonlinearity, but it is possible to approximate sampling invariance with arbitrary accuracy at the expense of additional computational cost. In this paper, we study morphological filters with line segments as structuring elements. We present a comparison of three known and three new methods to implement these filters. The method that yields a good compromise between accuracy and computational cost employs a (subpixel) skew to the image, followed by filtering along the grid axes using a discrete line segment, followed by an inverse skew. The staircase approximations to line segments under random orientations can be modeled by skewing a horizontal or vertical line segment. Rather than skewing the binary line segment we skew the image data, which substantially reduces quantization error. We proceed to determine the optimal number of orientations to use when measuring the length of line segments with unknown orientation.

Segmentation and size measurement of polyps in CT colonography.

Virtual colonoscopy is a relatively new method for the detection of colonic polyps. Their size, which is measured from reformatted CT images, mainly determines diagnosis. We present an automatic method for measuring the polyp size. The method is based on a robust segmentation method that grows a surface patch over the entire polyp surface starting from a seed. Projection of the patch points along the polyp axis yields a 2D point set to which we fit an ellipse. The long axis of the ellipse denotes the size of the polyp. We evaluate our method by comparing the automated size measurement with those of two radiologists using scans of a colon phantom. We give data for inter-observer and intra-observer variability of radiologists and our method as well as the accuracy and precision.

Alignment of the cell nucleus from labeled proteins only for 4D in vivo imaging.

Studies of protein dynamics by 4D (3D + time) confocal microscopy in vivo are hampered by global cell motion. The time between the acquisitions of the 3D images is in the order of minutes. Therefore, it is not to be expected that the cell as a whole remains fixed in the water basin on the stage. This superimposes a motion on the protein dynamics that has to be removed. We present a robust registration technique to align the cell images that does not require the a priori establishment of point-to-point correspondences. Instead, it uses the distribution of the labeled proteins. After correction for the translation, the 3D rotation of the cell is estimated. A robust intrinsic body coordinate system is constructed via the inertia tensor from the intensity distribution. By combining basis transformation to this intrinsic coordinate system, we can calculated the rotation matrix in a conceptual and computational straightforward manner. We have evaluated the performance of this approach in three experiments with human osteaosarcoma cells (U-2 OS), where the nuclear proteins Histon H4 and PML were visualized. The PML is concentrated in several dozen nuclear spots. Expression of Histon H4 results in a total nuclear staining. The registration results for both channels computed independently are very similar. Practically, this means that only the labeled material needs to be observed and still registration of the cell as a whole can be achieved.

Diffusion of microspheres in sealed and open microarrays.

In several experiments, we study the diffusion of microspheres with different radii in microarrays filled with a variety of aqueous solutions of ethylene glycol. We study diffusion in open and closed (sealed) microarrays. In sealed nanoliter wells, the tracers show pure diffusion, whereas in open reactors, a radial outward-directed evaporation-induced liquid flow is superimposed onto the diffusion. In general, one of the following quantities can be calculated if the others are known: the temperature, the viscosity of the medium, the radius of the microbeads, or the diffusion constant. The estimated diffusion constants in closed microarrays are in good agreement with theoretical predictions based on the Brownian motion. We monitor the motion of the microbeads under a microscope and extract their paths in time from the digital recordings. Ambiguous paths due to the crossing of two trajectories can be detected. We show that low microsphere concentrations or high viscosities do not hamper a robust estimation of the diffusion parameters.

Monitoring enzymatic reactions in nanolitre wells.

We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory-on-a-chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.

Ring formation in nanoliter cups: quantitative measurements of flow in micromachined wells.

Drying of DNA spots on microarrays and spilled coffee yields ringlike stains, because the outward flow transports dissolved particles to the border. Contact line pinning and diffusion limited evaporation of a liquid sample are the two necessary conditions to induce an outward directed liquid flow during evaporation. In this paper we present quantitative measurements of this flow field visualized by microspheres which are injected into a liquid sample in circular wells with a radius of 100-150 micro m and a depth of 6 micro m. The motion, including Brownian motion, of these microspheres with a radius of 0.25 micro m is recorded using digital fluorescence microscopy. Our analysis, using optic flow, does not require object identification, nor tracking of the individual objects. The spatiotemporal measurement space is sparsely filled at only those space/time positions where a microsphere is present. A confidence measure is computed indicating the presence of microspheres in this measurement space. The circular well shape allows us to transform the sparse measurement space into a denser, averaged radial velocity field. In this transformation we "interpolate" the radial velocity between values with a high confidence, which results in quantitative measurements of this outward flow field during the complete time interval of the evaporation process and at all radial positions in the circular wells. This allows for a quantitative validation of the elegant theory of ring formation.

Temporal phase-unwrapping algorithm for dynamic interference pattern analysis in interference-contrast microscopy.

A temporal phase-unwrapping algorithm has been developed for the analysis of dynamic interference patterns generated with interference-contrast microscopy in micromachined picoliter vials. These vials are etched in silicon dioxide, have a typical depth of 6 mum, and are filled with a liquid sample. In this kind of microscopy, fringe patterns are observed at the air-liquid interface. These fringe patterns are caused by interference between the directly reflected part of an incident plane wave and the part of that wave that is reflected on the bottom of the vial. The optical path difference (OPD) between both parts is proportional to the distance to the reflecting bottom of the vial. Evaporation decreases the OPD at the meniscus level and causes alternating constructive and destructive interference of the incident light, resulting in an interferogram. Imaging of the space-varying OPD yields a fringe pattern in which the isophotes correspond to isoheight curves of the meniscus. When the bottom is flat, the interference pattern allows for monitoring of the meniscus as a function of time during evaporation. However, when there are objects on the bottom of the vial, the heights of these objects are observed as phase jumps in the fringes proportional to their heights. First, we present a classical electromagnetic description of interference-contrast microscopy. Second, a temporal phase-unwrapping algorithm is described that retrieves the meniscus profile from the interference pattern. Finally, this algorithm is applied to measure height differences of objects on the bottom in other micromachined vials with a precision of ~5 nm.

Mean and variance of ratio estimators used in fluorescence ratio imaging.

BACKGROUND: The ratio of two measured fluorescence signals (called x and y) is used in different applications in fluorescence microscopy. Multiple instances of both signals can be combined in different ways to construct different ratio estimators. METHODS: The mean and variance of three estimators for the ratio between two random variables, x and y, are discussed. Given n samples of x and y, we can intuitively construct two different estimators: the mean of the ratio of each x and y and the ratio between the mean of x and the mean of y. The former is biased and the latter is only asymptotically unbiased. Using the statistical characteristics of this estimator, a third, unbiased estimator can be constructed. RESULTS: We tested the three estimators on simulated data, real-world fluorescence test images, and comparative genome hybridization (CGH) data. The results on the simulated and real-world test images confirm the presented theory. The CGH experiments show that our new estimator performs better than the existing estimators. CONCLUSIONS: We have derived an unbiased ratio estimator that outperforms intuitive ratio estimators.

Background estimation in nonlinear image restoration.

One of the essential ways in which nonlinear image restoration algorithms differ from linear, convolution-type image restoration filters is their capability to restrict the restoration result to nonnegative intensities. The iterative constrained Tikhonov-Miller (ICTM) algorithm, for example, incorporates the nonnegativity constraint by clipping all negative values to zero after each iteration. This constraint will be effective only when the restored intensities have near-zero values. Therefore the background estimation will have an influence on the effectiveness of the nonnegativity constraint of these algorithms. We investigated quantitatively the dependency of the performance of the ICTM, Carrington, and Richardson-Lucy algorithms on the estimation of the background and compared it with the performance of the linear Tikhonov-Miller restoration filter. We found that the performance depends critically on the background estimation: An underestimation of the background will make the nonnegativity constraint ineffective, which results in a performance that does not differ much from the Tikhonov-Miller filter performance. A (small) overestimation, however, degrades the performance dramatically, since it results in a clipping of object intensities. We propose a novel general method to estimate the background based on the dependency of nonlinear restoration algorithms on the background, and we demonstrate its applicability on real confocal images.

FISH and chips: automation of fluorescent dot counting in interphase cell nuclei.

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low. Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming. We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus. This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus. After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd., Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc., Cupertino, CA, USA) computer. After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images. The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome. The slides contained lymphocytes from cultured blood. We compared the results of the dot counter with manual counting. Manually obtained results, published in the literature, were used as the "ground truth." For a normal specimen, 97.5% of cells will have two dots. Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly. In other words, an average of 11% has to be interactively corrected, using a monitor display. The machine accuracies, after interactive correction, are comparable to panels of human experts (manual). The fully automatically obtained results are biased with respect to manual counting. An error analysis is given, and different causes are discussed.

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope.

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple means.

The Athena semi-automated karyotyping system.

In this article we describe Athena, a system that provides for semi-automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris-convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well-accepted Macintosh user interface.