Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Optimizing geometric accuracy of four dimensional CT scans acquired using the wall and couch mounted Varian Real-Time Position Management camera systems

Objective:

The aim of this study was to identify sources of anatomical misrepresentation due to the location of camera mounting, tumour motion velocity and image processing artefacts in order to optimise the 4DCT scan protocol and improve geometrical-temporal accuracy.

A phantom with an imaging insert was driven with a sinusoidal superior-inferior motion of varying amplitude and period for 4DCT scanning. The length of a high density cube within the insert was measured using treatment planning software to determine the accuracy of its spatial representation. Scan parameters were varied including the tube rotation period and the cine time between reconstructed images. A CT image quality phantom was used to measure various image quality signatures under the scan parameters tested.

No significant difference in spatial accuracy was found for 4DCT scans carried out using the wall mounted or couch mounted camera for sinusoidal target motion. Greater spatial accuracy was found for 4DCT scans carried out using a tube rotation speed of 0.5s rather than 1.0s. The reduction in image quality when using a faster rotation speed was not enough to require an increase in patient dose.

4DCT accuracy may be increased by optimising scan parameters, including choosing faster tube rotation speeds. Peak misidentification in the recorded breathing trace leads to spatial artefacts and this risk can be reduced by using a couch mounted infrared camera.

This study explicitly shows that 4DCT scan accuracy is improved by scanning with a faster CT tube rotation speed.

Quantitative ultrasound imaging during shear wave propagation for application related to breast cancer diagnosis

Dans le contexte de la caractérisation des tissus mammaires, on peut se demander ce que l’examen d’un attribut en échographie quantitative (« quantitative ultrasound » - QUS) d’un milieu diffusant (tel un tissu biologique mou) pendant la propagation d’une onde de cisaillement ajoute à son pouvoir discriminant. Ce travail présente une étude du comportement variable temporel de trois paramètres statistiques (l’intensité moyenne, le paramètre de structure et le paramètre de regroupement des diffuseurs) d’un modèle général pour l’enveloppe écho de l’onde ultrasonore rétrodiffusée (c.-à-d., la K-distribution homodyne) sous la propagation des ondes de cisaillement. Des ondes de cisaillement transitoires ont été générés en utilisant la mèthode d’ imagerie de cisaillement supersonique ( «supersonic shear imaging » - SSI) dans trois fantômes in-vitro macroscopiquement homogènes imitant le sein avec des propriétés mécaniques différentes, et deux fantômes ex-vivo hétérogénes avec tumeurs de souris incluses dans un milieu environnant d’agargélatine. Une comparaison de l’étendue des trois paramètres de la K-distribution homodyne avec et sans propagation d’ondes de cisaillement a montré que les paramètres étaient significativement (p < 0,001) affectès par la propagation d’ondes de cisaillement dans les expériences in-vitro et ex-vivo. Les résultats ont également démontré que la plage dynamique des paramétres statistiques au cours de la propagation des ondes de cisaillement peut aider à discriminer (avec p < 0,001) les trois fantômes homogènes in-vitro les uns des autres, ainsi que les tumeurs de souris de leur milieu environnant dans les fantômes hétérogénes ex-vivo. De plus, un modéle de régression linéaire a été appliqué pour corréler la plage de l’intensité moyenne sous la propagation des ondes de cisaillement avec l’amplitude maximale de déplacement du « speckle » ultrasonore. La régression linéaire obtenue a été significative : fantômes in vitro : R2 = 0.98, p < 0,001 ; tumeurs ex-vivo : R2 = 0,56, p = 0,013 ; milieu environnant ex-vivo : R2 = 0,59, p = 0,009. En revanche, la régression linéaire n’a pas été aussi significative entre l’intensité moyenne sans propagation d’ondes de cisaillement et les propriétés mécaniques du milieu : fantômes in vitro : R2 = 0,07, p = 0,328, tumeurs ex-vivo : R2 = 0,55, p = 0,022 ; milieu environnant ex-vivo : R2 = 0,45, p = 0,047. Cette nouvelle approche peut fournir des informations supplémentaires à l’échographie quantitative statistique traditionnellement réalisée dans un cadre statique (c.-à-d., sans propagation d’ondes de cisaillement), par exemple, dans le contexte de l’imagerie ultrasonore en vue de la classification du cancer du sein.

Parallel pipelined array architectures for real-time histogram computation in consumer devices

The real-time parallel computation of histograms using an array of pipelined cells is proposed and prototyped in this paper with application to consumer imaging products. The array operates in two modes: histogram computation and histogram reading. The proposed parallel computation method does not use any memory blocks. The resulting histogram bins can be stored into an external memory block in a pipelined fashion for subsequent reading or streaming of the results. The array of cells can be tuned to accommodate the required data path width in a VLSI image processing engine as present in many imaging consumer devices. Synthesis of the architectures presented in this paper in FPGA are shown to compute the real-time histogram of images streamed at over 36 megapixels at 30 frames/s by processing in parallel 1, 2 or 4 pixels per clock cycle.

ATD: a multiplatform for semiautomatic 3-D detection of kidneys and their pathology in real time

This research presents a novel multi-functional system for medical Imaging-enabled Assistive Diagnosis (IAD). Although the IAD demonstrator has focused on abdominal images and supports the clinical diagnosis of kidneys using CT/MRI imaging, it can be adapted to work on image delineation, annotation and 3D real-size volumetric modelling of other organ structures such as the brain, spine, etc. The IAD provides advanced real-time 3D visualisation and measurements with fully automated functionalities as developed in two stages. In the first stage, via the clinically driven user interface, specialist clinicians use CT/MRI imaging datasets to accurately delineate and annotate the kidneys and their possible abnormalities, thus creating “3D Golden Standard Models”. Based on these models, in the second stage, clinical support staff i.e. medical technicians interactively define model-based rules and parameters for the integrated “Automatic Recognition Framework” to achieve results which are closest to that of the clinicians. These specific rules and parameters are stored in “Templates” and can later be used by any clinician to automatically identify organ structures i.e. kidneys and their possible abnormalities. The system also supports the transmission of these “Templates” to another expert for a second opinion. A 3D model of the body, the organs and their possible pathology with real metrics is also integrated. The automatic functionality was tested on eleven MRI datasets (comprising of 286 images) and the 3D models were validated by comparing them with the metrics from the corresponding “3D Golden Standard Models”. The system provides metrics for the evaluation of the results, in terms of Accuracy, Precision, Sensitivity, Specificity and Dice Similarity Coefficient (DSC) so as to enable benchmarking of its performance. The first IAD prototype has produced promising results as its performance accuracy based on the most widely deployed evaluation metric, DSC, yields 97% for the recognition of kidneys and 96% for their abnormalities; whilst across all the above evaluation metrics its performance ranges between 96% and 100%. Further development of the IAD system is in progress to extend and evaluate its clinical diagnostic support capability through development and integration of additional algorithms to offer fully computer-aided identification of other organs and their abnormalities based on CT/MRI/Ultra-sound Imaging.

Reduced-order modeling of light transport in tissue for real-time monitoring of brain hemodynamics using diffuse optical tomography

This paper proposes a new reconstruction method for diffuse optical tomography using reduced-order models of light transport in tissue. The models, which directly map optical tissue parameters to optical flux measurements at the detector locations, are derived based on data generated by numerical simulation of a reference model. The reconstruction algorithm based on the reduced-order models is a few orders of magnitude faster than the one based on a finite element approximation on a fine mesh incorporating a priori anatomical information acquired by magnetic resonance imaging. We demonstrate the accuracy and speed of the approach using a phantom experiment and through numerical simulation of brain activation in a rat's head. The applicability of the approach for real-time monitoring of brain hemodynamics is demonstrated through a hypercapnic experiment. We show that our results agree with the expected physiological changes and with results of a similar experimental study. However, by using our approach, a three-dimensional tomographic reconstruction can be performed in ∼3  s per time point instead of the 1 to 2 h it takes when using the conventional finite element modeling approach

In-situ and real time scanning probe microscopy of organic ultra thin films

In recent decades, Organic Thin Film Transistors (OTFTs) have attracted lots of interest due to their low cost, large area and flexible properties which have brought them to be considered the building blocks of the future organic electronics. Experimentally, devices based on the same organic material deposited in different ways, i.e. by varying the deposition rate of the molecules, show different electrical performance. As predicted theoretically, this is due to the speed and rate by which charge carriers can be transported by hopping in organic thin films, transport that depends on the molecular arrangement of the molecules. This strongly suggests a correlation between the morphology of the organic semiconductor and the performance of the OTFT and hence motivated us to carry out an in-situ real time SPM study of organic semiconductor growth as an almost unprecedent experiment with the aim to fully describe the morphological evolution of the ultra-thin film and find the relevant morphological parameters affecting the OTFT electrical response. For the case of 6T on silicon oxide, we have shown that the growth mechanism is 2D+3D, with a roughening transition at the third layer and a rapid roughening. Relevant morphological parameters have been extracted by the AFM images. We also developed an original mathematical model to estimate theoretically and more accurately than before, the capacitance of an EFM tip in front of a metallic substrate. Finally, we obtained Ultra High Vacuum (UHV) AFM images of 6T at lying molecules layer both on silicon oxide and on top of 6T islands. Moreover, we performed ex-situ AFM imaging on a bilayer film composed of pentacene (a p-type semiconductor) and C60 (an n-type semiconductor).

Prospective navigator-echo-based real-time triggering of fetal head movement for the reduction of artifacts

The purpose of this study was to evaluate the neuroimaging quality and accuracy of prospective real-time navigator-echo acquisition correction versus untriggered intrauterine magnetic resonance imaging (MRI) techniques. Twenty women in whom fetal motion artifacts compromised the neuroimaging quality of fetal MRI taken during the 28.7 +/- 4 week of pregnancy below diagnostic levels were additionally investigated using a navigator-triggered half-Fourier acquired single-shot turbo-spin echo (HASTE) sequence. Imaging quality was evaluated by two blinded readers applying a rating scale from 1 (not diagnostic) to 5 (excellent). Diagnostic criteria included depiction of the germinal matrix, grey and white matter, CSF, brain stem and cerebellum. Signal-difference-to-noise ratios (SDNRs) in the white matter and germinal zone were quantitatively evaluated. Imaging quality improved in 18/20 patients using the navigator echo technique (2.4 +/- 0.58 vs. 3.65 +/- 0.73 SD, p < 0.01 for all evaluation criteria). In 2/20 patients fetal movement severely impaired image quality in conventional and navigated HASTE. Navigator-echo imaging revealed additional structural brain abnormalities and confirmed diagnosis in 8/20 patients. The accuracy improved from 50% to 90%. Average SDNR increased from 0.7 +/- 7.27 to 19.83 +/- 15.71 (p < 0.01). Navigator-echo-based real-time triggering of fetal head movement is a reliable technique that can deliver diagnostic fetal MR image quality despite vigorous fetal movement.

Real-time ultra-high optical coherence tomography monitoring of optical tissue effects caused by selective retina therapy

Purpose: Selective retina therapy (SRT) has shown great promise compared to conventional retinal laser photocoagulation as it avoids collateral damage and selectively targets the retinal pigment epithelium (RPE). Its use, however, is challenging in terms of therapy monitoring and dosage because an immediate tissue reaction is not biomicroscopically discernibel. To overcome these limitations, real-time optical coherence tomography (OCT) might be useful to monitor retinal tissue during laser application. We have thus evaluated a proprietary OCT system for its capability of mapping optical changes introduced by SRT in retinal tissue. Methods: Freshly enucleated porcine eyes, covered in DMEM upon collection were utilized and a total of 175 scans from ex-vivo porcine eyes were analyzed. The porcine eyes were used as an ex-vivo model and results compared to two time-resolved OCT scans, recorded from a patient undergoing SRT treatment (SRT Vario, Medical Laser Center Lübeck). In addition to OCT, fluorescin angiography and fundus photography were performed on the patient and OCT scans were subsequently investigated for optical tissue changes linked to laser application. Results: Biomicroscopically invisible SRT lesions were detectable in OCT by changes in the RPE / Bruch's complex both in vivo and the porcine ex-vivo model. Laser application produced clearly visible optical effects such as hyperreflectivity and tissue distortion in the treated retina. Tissue effects were even discernible in time-resolved OCT imaging when no hyper-reflectivity persisted after treatment. Data from ex-vivo porcine eyes showed similar to identical optical changes while effects visible in OCT appeared to correlate with applied pulse energy, leading to an additional reflective layer when lesions became visible in indirect ophthalmoscopy. Conclusions: Our results support the hypothesis that real-time high-resolution OCT may be a promising modality to obtain additional information about the extent of tissue damage caused by SRT treatment. Data shows that our exvivo porcine model adequately reproduces the effects occurring in-vivo, and thus can be used to further investigate this promising imaging technique.

Time-Resolved Ultra–High Resolution Optical Coherence Tomography for Real-Time Monitoring of Selective Retina Therapy

Purpose: Selective retina therapy (SRT) is a novel treatment for retinal pathologies, solely targeting the retinal pigment epithelium (RPE). During SRT, the detection of an immediate tissue reaction is challenging as tissue effects remain limited to intracellular RPE photodisruption. Time-resolved ultra-high axial resolution optical coherence tomography (OCT) is thus evaluated for the monitoring of dynamic optical changes at and around the RPE during SRT. Methods: An experimental OCT system with an ultra-high axial resolution of 1.78 µm was combined with an SRT system and time-resolved OCT M-scans of the target area were recorded from four patients undergoing SRT. OCT scans were analyzed and OCT morphology was correlated with findings in fluorescein angiography, fundus photography and cross-sectional OCT. Results: In cases where the irradiation caused RPE damage proven by fluorescein angiography, the lesions were well discernible in time-resolved OCT images but remained invisible in fundus photography and cross-sectional OCT acquired after treatment. If RPE damage was introduced, all applied SRT pulses led to detectable signal changes in the time-resolved OCT images. The extent of optical signal variation seen in the OCT data appeared to scale with the applied SRT pulse energy. Conclusion: The first clinical results proved that successful SRT irradiation induces detectable changes in the OCT M-scan signal while it remains invisible in conventional ophthalmoscopic imaging. Thus, real-time high-resolution OCT is a promising modality to monitor and analyze tissue effects introduced by selective retina therapy and may be used to guide SRT in an automatic feedback mode.

Real-Time In Vivo Characterization of Primary Liver Tumors With Diffuse Optical Spectroscopy During Percutaneous Needle Interventions: Feasibility Study in Woodchucks

OBJECTIVE This study presents the first in vivo real-time optical tissue characterization during image-guided percutaneous intervention using near-infrared diffuse optical spectroscopy sensing at the tip of a needle. The goal of this study was to indicate transition boundaries from healthy tissue to tumors, namely, hepatic carcinoma, based on the real-time feedback derived from the optical measurements. MATERIALS AND METHODS Five woodchucks with hepatic carcinoma were used for this study. The woodchucks were imaged with contrast-enhanced cone beam computed tomography with a flat panel detector C-arm system to visualize the carcinoma in the liver. In each animal, 3 insertions were performed, starting from the skin surface toward the hepatic carcinoma under image guidance. In 2 woodchucks, each end point of the insertion was confirmed with pathologic examination of a biopsy sample. While advancing the needle in the animals under image guidance such as fluoroscopy overlaid with cone beam computed tomography slice and ultrasound, optical spectra were acquired at the distal end of the needles. Optical tissue characterization was determined by translating the acquired optical spectra into clinical parameters such as blood, water, lipid, and bile fractions; tissue oxygenation levels; and scattering amplitude related to tissue density. The Kruskal-Wallis test was used to study the difference in the derived clinical parameters from the measurements performed within the healthy tissue and the hepatic carcinoma. Kurtoses were calculated to assess the dispersion of these parameters within the healthy and carcinoma tissues. RESULTS Blood and lipid volume fractions as well as tissue oxygenation and reduced scattering amplitude showed to be significantly different between the healthy part of the liver and the hepatic carcinoma (P < 0.05) being higher in normal liver tissue. A decrease in blood and lipid volume fractions and tissue oxygenation as well as an increase in scattering amplitude were observed when the tip of the needle crossed the margin from the healthy liver tissue to the carcinoma. The kurtosis for each derived clinical parameter was high in the hepatic tumor as compared with that in the healthy liver indicating intracarcinoma variability. CONCLUSIONS Tissue blood content, oxygenation level, lipid content, and tissue density all showed significant differences when the needle tip was guided from the healthy tissue to the carcinoma and can therefore be used to identify tissue boundaries during percutaneous image-guided interventions.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Reproducibility and accuracy of echocardiographic measurements of left ventricular parameters using real-time three-dimensional echocardiography

OBJECTIVES We sought to determine whether assessment of left ventricular (LV) function with real-time (RT) three-dimensional echocardiography (3DE) could reduce the variation of sequential LV measurements and provide greater accuracy than two-dimensional echocardiography (2DE). BACKGROUND Real-time 3DE has become feasible as a standard clinical tool, but its accuracy for LV assessment has not been validated. METHODS Unselected patients (n = 50; 41 men; age, 64 +/- 8 years) presenting for evaluation of LV function were studied with 2DE and RT-3DE. Test-retest variation was performed by a complete restudy by a separate sonographer within 1 h without alteration of hemodynamics or therapy. Magnetic resonance imaging (MRI) images were obtained during a breath-hold, and measurements were made off-line. RESULTS The test-retest variation showed similar measurements for volumes but wider scatter of LV mass measurements with M-mode and 2DE than 3DE. The average MRI end-diastolic volume was 172 +/- 53 ml; LV volumes were underestimated by 2DE (mean difference, -54 +/- 33; p < 0.01) but only slightly by RT-3DE (-4 +/- 29; p = 0.31). Similarly, end-systolic volume by MRI (91 +/- 53 ml) was underestimated by 2DE (mean difference, -28 +/- 28; p < 0.01) and by RT-3DE (mean difference, -3 +/- 18; p = 0.23). Ejection fraction by MRI was similar by 2DE (p = 0.76) and RT-3DE (p = 0.74). Left ventricular mass (183 +/- 50 g) was overestimated by M-mode (mean difference, 68 +/- 86 g; p < 0.01) and 2DE (16 +/- 57; p = 0.04) but not RT-3DE (0 +/- 38 g; p = 0.94). There was good inter- and intra-observer correlation between RT-3DE by two sonographers for volumes, ejection fraction, and mass. CONCLUSIONS Real-time 3DE is a feasible approach to reduce test-retest variation of LV volume, ejection fraction, and mass measurements in follow-up LV assessment in daily practice. (C) 2004 by the American College of Cardiology Foundation.

Use of real-time three-dimensional echocardiography to measure left atrial volume: Comparison with other echocardiographic techniques

Objectives: Left atrial (LA) volume (LAV) is a prognostically important biomarker for diastolic dysfunction, but its reproducibility on repeated testing is not well defined. LA assessment with 3-dimensional. (3D) echocardiography (3DE) has been validated against magnetic resonance imaging, and we sought to assess whether this was superior to existing measurements for sequential echocardiographic follow-up. Methods: Patients (n = 100; 81 men; age 56 +/- 14 years) presenting for LA evaluation were studied with M-mode (MM) echocardiography, 2-dimensional (2D) echocardiography, and 3DE. Test-retest variation was performed by a complete restudy by a separate sonographer within 1 hour without alteration of hemodynamics or therapy. In all, 20 patients were studied for interobserver and intraobserver variation. LAVs were calculated by using M-mode diameter and planimetered atrial area in the apical. 4-chamber view to calculate an assumed sphere, as were prolate ellipsoid, Simpson's biplane, and biplane area-length methods. All were compared with 3DE. Results: The average LAV was 72 +/- 27 mL by 3DE. There was significant underestimation of LAV by M-mode (35 +/- 20 mL, r = 0.66, P < .01). The 3DE and various 2D echocardiographic techniques were well correlated: LA planimetry (85 +/- 38 mL, r = 0.77, P < .01), prolate ellipsoid (73 +/- 36 mL, r = 0.73, P = .04), area-length (64 +/- 30 mL, r = 0.74, P < .01), and Simpson's biplane (69 +/- 31 mL, r = 0.78, P = .06). Test-retest variation for 3DE was most favorable (r = 0.98, P < .01), with the prolate ellipsoid method showing most variation. Interobserver agreement between measurements was best for 3DE (r = 0.99, P < .01), with M-mode the worst (r = 0.89, P < .01). Intraobserver results were similar to interobserver, the best correlation for 3DE (r = 0.99, P < .01), with LA planimetry the worst (r = 0.91, P < .01). Conclusions. The 2D measurements correlate closely with 3DE. Follow-up assessment in daily practice appears feasible and reliable with both 2D and 3D approaches.

Design, analysis and evaluation of sigma-delta based beamformers for medical ultrasound imaging applications

The inherent analogue nature of medical ultrasound signals in conjunction with the abundant merits provided by digital image acquisition, together with the increasing use of relatively simple front-end circuitries, have created considerable demand for single-bit  beamformers in digital ultrasound imaging systems. Furthermore, the increasing need to design lightweight ultrasound systems with low power consumption and low noise, provide ample justification for development and innovation in the use of single-bit  beamformers in ultrasound imaging systems. The overall aim of this research program is to investigate, establish, develop and confirm through a combination of theoretical analysis and detailed simulations, that utilize raw phantom data sets, suitable techniques for the design of simple-to-implement hardware efficient  digital ultrasound beamformers to address the requirements for 3D scanners with large channel counts, as well as portable and lightweight ultrasound scanners for point-of-care applications and intravascular imaging systems. In addition, the stability boundaries of higher-order High-Pass (HP) and Band-Pass (BP) Σ−Δ modulators for single- and dual- sinusoidal inputs are determined using quasi-linear modeling together with the describing-function method, to more accurately model the  modulator quantizer. The theoretical results are shown to be in good agreement with the simulation results for a variety of input amplitudes, bandwidths, and modulator orders. The proposed mathematical models of the quantizer will immensely help speed up the design of higher order HP and BP Σ−Δ modulators to be applicable for digital ultrasound beamformers. Finally, a user friendly design and performance evaluation tool for LP, BP and HP  modulators is developed. This toolbox, which uses various design methodologies and covers an assortment of  modulators topologies, is intended to accelerate the design process and evaluation of  modulators. This design tool is further developed to enable the design, analysis and evaluation of  beamformer structures including the noise analyses of the final B-scan images. Thus, this tool will allow researchers and practitioners to design and verify different reconstruction filters and analyze the results directly on the B-scan ultrasound images thereby saving considerable time and effort.