Intracellular nanomanipulation by a photonic-force microscope with real-time acquisition of a 3D stiffness matrix.

A traditional photonic-force microscope (PFM) results in huge sets of data, which requires tedious numerical analysis. In this paper, we propose instead an analog signal processor to attain real-time capabilities while retaining the richness of the traditional PFM data. Our system is devoted to intracellular measurements and is fully interactive through the use of a haptic joystick. Using our specialized analog hardware along with a dedicated algorithm, we can extract the full 3D stiffness matrix of the optical trap in real time, including the off-diagonal cross-terms. Our system is also capable of simultaneously recording data for subsequent offline analysis. This allows us to check that a good correlation exists between the classical analysis of stiffness and our real-time measurements. We monitor the PFM beads using an optical microscope. The force-feedback mechanism of the haptic joystick helps us in interactively guiding the bead inside living cells and collecting information from its (possibly anisotropic) environment. The instantaneous stiffness measurements are also displayed in real time on a graphical user interface. The whole system has been built and is operational; here we present early results that confirm the consistency of the real-time measurements with offline computations.

Quantitative morphometrical characterization of human pronuclear zygotes.

BACKGROUND: Identification of embryos with high implantation potential remains a challenge in in vitro fertilization (IVF). Subjective pronuclear (PN) zygote scoring systems have been developed for that purpose. The aim of this work was to provide a software tool that enables objective measuring of morphological characteristics of the human PN zygote. METHODS: A computer program was created to analyse zygote images semi-automatically, providing precise morphological measurements. The accuracy of this approach was first validated by comparing zygotes from two different IVF centres with computer-assisted measurements or subjective scoring. Computer-assisted measurement and subjective scoring were then compared for their ability to classify zygotes with high and low implantation probability by using a linear discriminant analysis. RESULTS: Zygote images coming from the two IVF centres were analysed with the software, resulting in a series of precise measurements of 24 variables. Using subjective scoring, the cytoplasmic halo was the only feature which was significantly different between the two IVF centres. Computer-assisted measurements revealed significant differences between centres in PN centring, PN proximity, cytoplasmic halo and features related to nucleolar precursor bodies distribution. The zygote classification error achieved with the computer-assisted measurements (0.363) was slightly inferior to that of the subjective ones (0.393). CONCLUSIONS: A precise and objective characterization of the morphology of human PN zygotes can be achieved by the use of an advanced image analysis tool. This computer-assisted analysis allows for a better morphological characterization of human zygotes and can be used for classification.

Snakuscules.

A snakuscule (a minuscule snake) is the simplest active contour that we were able to design while keeping the quintessence of traditional snakes: an energy term governed by the data, and a regularization term. Our construction is an area-based snake, as opposed to curve-based snakes. It is parameterized by just two points, thus further easing requirements on the optimizer. Despite their ultimate simplicity, snakuscules retain enough versatility to be employed for solving various problems such as cell counting and segmentation of approximately circular features. In this paper, we detail the design process of a snakuscule and illustrate its usefulness through practical examples. We claim that our didactic intentions are well served by the simplicity of snakuscules.

Spline-based image-to-volume registration for three-dimensional electron microscopy.

This paper presents an algorithm based on a continuous framework for a posteriori angular and translational assignment in three-dimensional electron microscopy (3DEM) of single particles. Our algorithm can be used advantageously to refine the assignment of standard quantized-parameter methods by registering the images to a reference 3D particle model. We achieve the registration by employing a gradient-based iterative minimization of a least-squares measure of dissimilarity between an image and a projection of the volume in the Fourier transform (FT) domain. We compute the FT of the projection using the central-slice theorem (CST). To compute the gradient accurately, we take advantage of a cubic B-spline model of the data in the frequency domain. To improve the robustness of the algorithm, we weight the cost function in the FT domain and apply a "mixed" strategy for the assignment based on the minimum value of the cost function at registration for several different initializations. We validate our algorithm in a fully controlled simulation environment. We show that the mixed strategy improves the assignment accuracy; on our data, the quality of the angular and translational assignment was better than 2 voxel (i.e., 6.54 angstroms). We also test the performance of our algorithm on real EM data. We conclude that our algorithm outperforms a standard projection-matching refinement in terms of both consistency of 3D reconstructions and speed.

Chronic inhalation study of fiber glass and amosite asbestos in hamsters: twelve-month preliminary results.

The effects of chronic inhalation of glass fibers and amosite asbestos are currently under study in hamsters. The study includes 18 months of inhalation exposure followed by lifetime recovery. Syrian golden hamsters are exposed, nose only, for 6 hr/day, 5 day/week to size-selected test fibers: MMVF10a (Schuller 901 insulation glass); MMVF33 (Schuller 475 durable glass); amosite asbestos (three doses); or to filtered air (controls). Here we report interim results on airborne fiber characterization, lung fiber burden, and pathology (preliminary) through 12 months. Aerosolized test fibers averaged 15 to 20 microns in length and 0.5 to 1 micron in diameter. Target aerosol concentrations of World Health Organization (WHO) fibers (longer than 5 microns) were 250 fibers/cc for MMVF10a and MMVF33, and 25, 125, or 250 fibers/cc for amosite. WHO fiber lung burdens showed time-dependent and (for amosite) dose-dependent increases. After a 12-month exposure, lung burdens of fibers longer than 20 microns were greatest with amosite high and mid doses, similar for low-dose amosite and MMVF33, and smaller for MMVF10a. Biological responses of animals exposed for 12 months to MMVF10a were limited to nonspecific pulmonary inflammation. However, exposures to MMVF33 and each of three doses of amosite were associated with lung fibrosis and possible mesotheliomas (1 with MMVF33 and 2, 3, and 1 with amosite low, mid, and high doses, respectively). Pulmonary and pleural changes associated with amosite were qualitatively and quantitatively more severe than those associated with MMVF33. As of the 12-month time point, this study demonstrates that two different fiber glass compositions with similar fiber dimensions but different durabilities can have distinctly different effects on the hamster lung and pleura after inhalation exposure. (Preliminary tumor data through 18 months of exposure and 6 weeks of postexposure recovery became available as this manuscript went to press: No tumors were observed in the control or MMVF10a groups, and no additional tumors were observed in the MMVF33 group; however, a number of additional mesotheliomas were observed in the amosite groups.

Biopersistences of man-made vitreous fibers and crocidolite fibers in rat lungs following short-term exposures.

Biopersistence of commercial man-made vitreous fibers (MMVF) and crocidolite were studied in Fischer 344 rats. MMVF used were size-selected to be rat-respirable, and rats were exposed nose-only 6 h/day for 5 days to gravimetric concentrations (30 mg/m3) of two fiber glass compositions--a rockwool, and a slagwool--or to 10 mg/m3 of long-fibered crocidolite, or to filtered air. Animals were sacrificed at 1 hr, 1, 5, 31, 90, 180, 270, 365, and 545 days after exposure stopped. Fibers were recovered from digested lung tissue to determine changes in concentrations (fibers/mg dry lung) and fiber retentions (expressed as percent of day 1 retention [PR]) for selected dimension categories. One-day average concentrations of lung-retained MMVF and crocidolite fibers, of diameter > or = 0.5 micron or > 20 microns in length, were nearly equal, permitting direct comparisons between MMVF and crocidolite. At 270 days average PR for MMVF > or = 0.5 micron in diameter were from 3 to 6 +/- 2% and 27 +/- 9% for crocidolite. For fibers > 20 microns, PR were 1 to 4 +/- 4% for MMVF and 37 +/- 20% for crocidolite. At 545 days, MMVF > 20 microns in length were at background level while concentration of crocidolite fibers > 20 microns in length remained at 2000 +/- 400 f/mg DL (dry lung), or 38 +/- 9% of day-1 retention. These results suggest strongly that MMVF dissolved or fractured in vivo whereas crocidolite fibers did not change.

In situ microscopic analysis of asbestos and synthetic vitreous fibers retained in hamster lungs following inhalation.

Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.

Inhalation toxicology of decamethylcyclopentasiloxane (D5) following a 3-month nose-only exposure in Fischer 344 rats.

D5 is a low-molecular-weight cyclic siloxane used for industrial and consumer product applications. The objective of the present study was to evaluate the subchronic toxicity of D5 following a 3-month nose-only inhalation exposure. In addition, animals from both sexes of the control and high dose groups were allowed a 4-week recovery period to observe reversibility, persistence, or delayed occurrence of any potential adverse effects. Male and female Fischer 344 rats were exposed for 6 h/day, 5 days/week for 3 months to target concentrations of 0 (30/sex/group), 26 (20/sex/group), 46 (20/sex/group), 86 (20/sex/group), and 224 (30/sex/group) ppm D5. Recovery groups (0 and 224 ppm) comprised 10 rats/sex/group. Body weights and food consumption were monitored at least twice weekly over the course of exposures. Approximately 16 h preceding euthanasia, animals were transferred into metabolism caging for urine collection and were fasted. Rats were anesthetized with pentobarbital and euthanized by exsanguination. Blood was collected for hematological and clinical biochemical analyses. Selected organ weights were measured and a complete set of tissues was taken for histopathological examination. There were several minor changes observed in clinical biochemistry parameters; the most notable was an increase in gamma glutamyl transferase (gamma-GT) in both sexes at the high dose. In females, this effect was dose-related (46-224 ppm) and did not recover upon cessation of exposure. Additionally, there was an decrease in serum lactate dehydrogenase (LDH) observed in females at 86 and 224 ppm which was not resolved during recovery. There was an increase in absolute and/or relative liver weight in rats of both sexes. Taken together, these data suggest that the female rat is more sensitive to the actions of D5 on the liver. Exposure-related increases in absolute and relative lung weights were observed in both sexes at terminal necropsy. This observation was not noted in males in the recovery phase, but was still present in females. Finally, histopathological evidence indicated the primary target organ following D5 inhalation exposure is the lung, with an increase in focal macrophage accumulation and interstitial inflammation in the lungs of male and female rats exposed to 224 ppm D5. This observation did not appear to resolve at the end of a 1-month period of nonexposure. The incidence of these changes was also slightly increased in rats of both sexes exposed to 86 ppm D5. These data suggest that nose-only D5 vapor inhalation provokes minimal changes in the lung which are similar in incidence and severity to spontaneously occurring changes in control animals after nose-only exposures. There were no histopathological findings noted in the livers which support this organ as a target in this study, despite the observed changes in organ weight and in some serum chemistry parameters.

Distribution and attachment of Trypanosoma (Nannomonas) congolense in the proximal part of the proboscis of Glossina morsitans morsitans.

The distribution and attachment of Trypanosoma congolense were investigated in the proximal part of the proboscis of Glossina m. morsitans. In the food canal, epimastigotes and trypomastigotes formed tufts or compact layers. Trypanosomes were attached to the cuticle by their flagella, which formed zonar hemidesmosomes. The flagella were mostly attached parallel to the axis of the labrum and often pointed to its tip. Foot-like processes of the flagella came into contact with adjacent flagella leading to dense grouping of the trypanosomes. Despite narrow contacts between adjacent flagella, no desmosomes were differentiated. The trypanosomes were attached to all parts of the LCI sensilla and might thus impair their function. Uncoated and coated forms were present in the labrum, which indicated that transformation to metacyclic forms is not strictly limited to the hypopharynx. Uncoated forms were often attached to the cuticle of the common salivary duct and hypopharynx by hemidesmosomes. Coated forms could also be attached. Neither compact layers nor tufts of trypanosomes were found. This attachment may partly explain the low number of metacyclics deposited when flies probed on a warm glass slide.

Interpolation revisited.

Based on the theory of approximation, this paper presents a unified analysis of interpolation and resampling techniques. An important issue is the choice of adequate basis functions. We show that, contrary to the common belief, those that perform best are not interpolating. By opposition to traditional interpolation, we call their use generalized interpolation; they involve a prefiltering step when correctly applied. We explain why the approximation order inherent in any basis function is important to limit interpolation artifacts. The decomposition theorem states that any basis function endowed with approximation order can be expressed as the convolution of a B-spline of the same order with another function that has none. This motivates the use of splines and spline-based functions as a tunable way to keep artifacts in check without any significant cost penalty. We discuss implementation and performance issues, and we provide experimental evidence to support our claims.

Unwarping of unidirectionally distorted EPI images.

Echo-planar imaging (EPI) is a fast nuclear magnetic resonance imaging (MRI) method. Unfortunately, local magnetic field inhomogeneities induced mainly by the subject's presence cause significant geometrical distortion, predominantly along the phase-encoding direction, which must be undone to allow for meaningful further processing. So far, this aspect has been too often neglected. In this paper, we suggest a new approach using an algorithm specifically developed for the automatic registration of distorted EPI images with corresponding anatomically correct MRI images. We model the deformation field with splines, which gives us a great deal of flexibility, while comprising the affine transform as a special case. The registration criterion is least squares. Interestingly, the complexity of its evaluation does not depend on the resolution of the control grid. The spline model gives us good accuracy thanks to its high approximation order. The short support of splines leads to a fast algorithm. A multiresolution approach yields robustness and additional speedup. The algorithm was tested on real as well as synthetic data, and the results were compared with a manual method. A wavelet-based Sobolev-type random deformation generator was developed for testing purposes. A blind test indicates that the proposed automatic method is faster, more reliable, and more precise than the manual one.

Optimization of mutual information for multiresolution image registration.

We propose a new method for the intermodal registration of images using a criterion known as mutual information. Our main contribution is an optimizer that we specifically designed for this criterion. We show that this new optimizer is well adapted to a multiresolution approach because it typically converges in fewer criterion evaluations than other optimizers. We have built a multiresolution image pyramid, along with an interpolation process, an optimizer, and the criterion itself, around the unifying concept of spline-processing. This ensures coherence in the way we model data and yields good performance. We have tested our approach in a variety of experimental conditions and report excellent results. We claim an accuracy of about a hundredth of a pixel under ideal conditions. We are also robust since the accuracy is still about a tenth of a pixel under very noisy conditions. In addition, a blind evaluation of our results compares very favorably to the work of several other researchers.

Convolution-based interpolation for fast, high-quality rotation of images.

This paper focuses on the design of fast algorithms for rotating images and preserving high quality. The basis for the approach is a decomposition of a rotation into a sequence of one-dimensional translations. As the accuracy of these operations is critical, we introduce a general theoretical framework that addresses their design and performance. We also investigate the issue of optimality and present an improved least-square formulation of the problem. This approach leads to a separable three-pass implementation of a rotation using one-dimensional convolutions only. We provide explicit filter formulas for several continuous signal models including spline and bandlimited representations. Finally, we present rotation experiments and compare the currently standard techniques with the various versions of our algorithm. Our results indicate that the present algorithm in its higher-order versions outperforms all standard high-accuracy methods of which we are aware, both in terms of speed and quality. Its computational complexity increases linearly with the order of accuracy. The best-quality results are obtained with the sine-based algorithm, which can be implemented using simple one-dimensional FFTs.

MOMS: maximal-order interpolation of minimal support.

We consider the problem of interpolating a signal using a linear combination of shifted versions of a compactly-supported basis function phi(x). We first give the expression for the cases of phi's that have minimal support for a given accuracy (also known as "approximation order"). This class of functions, which we call maximal-order-minimal-support functions (MOMS) is made of linear combinations of the B-spline of the same order and of its derivatives. We provide an explicit form of the MOMS that maximizes the approximation accuracy when the step-size is small enough. We compute the sampling gain obtained by using these optimal basis functions over the splines of the same order. We show that it is already substantial for small orders and that it further increases with the approximation order L. When L is large, this sampling gain becomes linear; more specifically, its exact asymptotic expression is 2/(pie)L. Since the optimal functions are continuous, but not differentiable, for even orders, and even only piecewise continuous for odd orders, our result implies that regularity has little to do with approximating performance. These theoretical findings are corroborated by experimental evidence that involves compounded rotations of images.

A pyramid approach to subpixel registration based on intensity.

We present an automatic subpixel registration algorithm that minimizes the mean square intensity difference between a reference and a test data set, which can be either images (two-dimensional) or volumes (three-dimensional). It uses an explicit spline representation of the images in conjunction with spline processing, and is based on a coarse-to-fine iterative strategy (pyramid approach). The minimization is performed according to a new variation (ML*) of the Marquardt-Levenberg algorithm for nonlinear least-square optimization. The geometric deformation model is a global three-dimensional (3-D) affine transformation that can be optionally restricted to rigid-body motion (rotation and translation), combined with isometric scaling. It also includes an optional adjustment of image contrast differences. We obtain excellent results for the registration of intramodality positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) data. We conclude that the multiresolution refinement strategy is more robust than a comparable single-stage method, being less likely to be trapped into a false local optimum. In addition, our improved version of the Marquardt-Levenberg algorithm is faster.

Model-based estimation of 3-D stiffness parameters in photonic-force microscopy.

We propose a system to characterize the 3-D diffusion properties of the probing bead trapped by a photonic-force microscope. We follow a model-based approach, where the model of the dynamics of the bead is given by the Langevin equation. Our procedure combines software and analog hardware to measure the corresponding stiffness matrix. We are able to estimate all its elements in real time, including off-diagonal terms. To achieve our goal, we have built a simple analog computer that performs a continuous preprocessing of the data, which can be subsequently digitized at a much lower rate than is otherwise required. We also provide an effective numerical algorithm for compensating the correlation bias introduced by a quadrant photodiode detector in the microscope. We validate our approach using simulated data and show that our bias-compensation scheme effectively improves the accuracy of the system. Moreover, we perform experiments with the real system and demonstrate real-time capabilities. Finally, we suggest a simple adjunction that would allow one to determine the mass matrix as well.

Non-rigid consistent registration of 2D image sequences.

We present a novel algorithm for the registration of 2D image sequences that combines the principles of multiresolution B-spline-based elastic registration and those of bidirectional consistent registration. In our method, consecutive triples of images are iteratively registered to gradually extend the information through the set of images of the entire sequence. The intermediate results are reused for the registration of the following triple. We choose to interpolate the images and model the deformation fields using B-spline multiresolution pyramids. Novel boundary conditions are introduced to better characterize the deformations at the boundaries. In the experimental section, we quantitatively show that our method recovers from barrel/pincushion and fish-eye deformations with subpixel error. Moreover, it is more robust against outliers--occasional strong noise and large rotations--than the state-of-the-art methods. Finally, we show that our method can be used to realign series of histological serial sections, which are often heavily distorted due to folding and tearing of the tissues.

Myocardial perfusion assessment by use of system identification method in a one-compartment model.

Cardiovascular magnetic resonance has been shown to provide high data quality for myocardial perfusion assessment. However, to analyze the perfusion data, some signal processing and modeling is needed to correct for motion related artifacts and limited spatial resolution. This study describes a method based on system identification, allowing, after a first step of image registration, to integrate and correct the partial volume effect in the myocardium perfusion quantification. This method is then applied to patients with coronary artery disease or hypertrophic obstructive cardiomyopathy.

An Optimized Spline-Based Registration of a 3D CT to a Set of C-Arm Images.

We have developed an algorithm for the rigid-body registration of a CT volume to a set of C-arm images. The algorithm uses a gradient-based iterative minimization of a least-squares measure of dissimilarity between the C-arm images and projections of the CT volume. To compute projections, we use a novel method for fast integration of the volume along rays. To improve robustness and speed, we take advantage of a coarse-to-fine processing of the volume/image pyramids. To compute the projections of the volume, the gradient of the dissimilarity measure, and the multiresolution data pyramids, we use a continuous image/volume model based on cubic B-splines, which ensures a high interpolation accuracy and a gradient of the dissimilarity measure that is well defined everywhere. We show the performance of our algorithm on a human spine phantom, where the true alignment is determined using a set of fiducial markers.

Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions.

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone.