Photophysics of Deoxycytidine and 5-Methyldeoxycytidine in Solution: A Comprehensive Picture by Quantum Mechanical Calculations and Femtosecond Fluorescence Spectroscopy.

The study concerns the relaxation of electronic excited states of the DNA nucleoside deoxycytidine (dCyd) and its methylated analogue 5-methyldeoxycytidine (5mdCyd), known to be involved in the formation of UV-induced lesions of the genetic code. Due to the existence of four closely lying and potentially coupled excited states, the deactivation pathways in these systems are particularly complex and have not been assessed so far. Here, we provide a complete mechanistic picture of the excited state relaxation of dCyd/5mdCyd in three solvents-water, acetonitrile, and tetrahydrofuran-by combining femtosecond fluorescence experiments, addressing the effect of solvent proticity on the relaxation dynamics of dCyd and 5mdCyd for the first time, and two complementary quantum mechanical approaches (CASPT2/MM and PCM/TD-CAM-B3LYP). The lowest energy ππ* state is responsible for the sub-picosecond lifetime observed for dCyd in all the solvents. In addition, computed excited state absorption and transient IR spectra allow one, for the first time, to assign the tens of picoseconds time constant, reported previously, to a dark state (nOπ*) involving the carbonyl lone pair. A second low-lying dark state, involving the nitrogen lone pair (nNπ*), does significantly participate in the excited state dynamics. The 267 nm excitation of dCyd leads to a non-negligible population of the second bright ππ* state, which affects the dynamics, acting mainly as a "doorway" state for the nOπ* state. The solvent plays a key role governing the interplay between the different excited states; unexpectedly, water favors population of the dark states. In the case of 5mdCyd, an energy barrier present on the main nonradiative decay route explains the 6-fold lengthening of the excited state lifetime compared to that of dCyd, observed for all the examined solvents. Moreover, C5-methylation destabilizes both nOπ* and nNπ* dark states, thus preventing them from being populated.

An automatic system for in vitro cell migration studies.

This paper describes a system for in vitro cell migration analysis. Adult neural stem/progenitor cells are studied using time-lapse bright-field microscopy and thereafter stained immunohistochemically to find and distinguish undifferentiated glial progenitor cells and cells having differentiated into type-1 or type-2 astrocytes. The cells are automatically segmented and tracked through the time-lapse sequence. An extension to the Chan-Vese Level Set segmentation algorithm, including two new terms for specialized growing and pruning, made it possible to resolve clustered cells, and reduced the tracking error by 65%. We used a custom-built manual correction module to form a ground truth used as a reference for tracked cells that could be identified from the fluorescence staining. On average, the tracks were correct 95% of the time, using our new segmentation. The tracking, or association of segmented cells, was performed using a 2-state Hidden Markov Model describing the random behaviour of the cells. By re-estimating the motion model to conform with the segmented data we managed to reduce the number of tracking parameters to essentially only one. Upon characterization of the cell migration by the HMM state occupation function, it was found that glial progenitor cells were moving randomly 2/3 of the time, while the type-2 astrocytes showed a directed movement 2/3 of the time. This finding indicates possibilities for cell-type specific identification and cell sorting of live cells based on specific movement patterns in individual cell populations, which would have valuable applications in neurobiological research.

Estradiol measurement after GnRH-stimulation as a method to diagnose the presence of ovaries in the female domestic cat.

There is currently no method to reliably diagnose the presence of ovarian tissue in inactive intact female cats except laparatomy which is an invasive procedure. All available tests require that the queen is in estrus. Obvious overt symptoms of estrus are, however, not always observed and some queens may have only 2 estrus periods/year. Therefore this study was designed to evaluate if it is possible to diagnose the presence of ovarian tissue by measurement of estradiol before and/or after stimulation with a GnRH-analogue. Twenty-two female cats were divided into two groups; 11 females that were known to have been ovariectomized and 11 females that were known to be intact. From each cat a heparinised blood sample was collected from the cephalic vein for resting estradiol and progesterone measurements. All cats were treated with a GnRH-analogue buserelin (Receptal, 0.4microg/kg im). Two hours later a second blood sample was collected. Median estradiol increased after stimulation with buserelin in intact but not in ovariectomized females (11 range 5-21 vs. 20 range 12-41, P=0.004 and 6 range 4-9 vs. 6 range 5-9 P=0.8) and did not overlap between the two groups. The highest estradiol concentration post-GnRH in the ovariectomized group was 9pmol/L while the lowest in the intact group was 12pmol/L. Progesterone was basal in all cats except one both before and after GnRH-stimulation. In conclusion this study demonstrates that measurement of estradiol concentration in plasma 2h after stimulation with a GnRH-analogue seems to be a reliable method to diagnose the presence of ovarian tissue in the female cat.

Rotational diffusion of the 7-diethylamino-4-methylcoumarin C1 dye molecule in polar protic and aprotic solvents.

Fluorescence anisotropy decays of the 7-diethylamino-4-methylcoumarin C1 in various polar solvents of different viscosities and hydrogen bond donor/acceptor character have been recorded by means of the fluorescence upconversion and time-correlated single photon counting techniques. The resulting characteristic times for the rotational diffusion fall into two classes with regards to the viscosity-dependency: n-alcohols and "other" solvents. This deviation from the simple Stokes-Einstein-Debye model may be interpreted in terms of rotation of the coumarin molecule under two different hydrodynamic boundary-conditions ("stick" or "slip") in the two solvent classes. Possible explanations for this behaviour are discussed, and in particular solvent attachment and additional dielectric friction. Both these phenomena may in fact, under certain conditions, explain our findings. Our opinion, however, is that the dielectric friction model offers a more realistic picture of the additional rotational friction experienced by C1 in n-alcohols.

Flame front tracking by laser induced fluorescence spectroscopy and advanced image analysis.

This paper presents advanced image analysis methods for extracting information from high speed Planar Laser Induced Fluorescence (PLIF) data obtained from turbulent flames. The application of non-linear anisotropic diffusion filtering and of Active Contour Models (Snakes) is described to isolate flame boundaries. In a subsequent step, the detected flame boundaries are tracked in time using a frequency domain contour interpolation scheme. The implementations of the methods are described and possible applications of the techniques are discussed.

A multiscale dynamic programming procedure for boundary detection in ultrasonic artery images.

Ultrasonic measurements of human carotid and femoral artery walls are conventionally obtained by manually tracing interfaces between tissue layers. The drawbacks of this method are the interobserver variability and inefficiency. In this paper, we present a new automated method which reduces these problems. By applying a multiscale dynamic programming (DP) algorithm, approximate vessel wall positions are first estimated in a coarse-scale image, which then guide the detection of the boundaries in a fine-scale image. In both cases, DP is used for finding a global optimum for a cost function. The cost function is a weighted sum of terms, in fuzzy expression forms, representing image features and geometrical characteristics of the vessel interfaces. The weights are adjusted by a training procedure using human expert tracings. Operator interventions, if needed, also take effect under the framework of global optimality. This reduces the amount of human intervention and, hence, variability due to subjectiveness. By incorporating human knowledge and experience, the algorithm becomes more robust. A thorough evaluation of the method in the clinical environment shows that interobserver variability is evidently decreased and so is the overall analysis time. We conclude that the automated procedure can replace the manual procedure and leads to an improved performance.

Compensation method for attenuated planar laser images of optically dense sprays.

We present a method for compensating laser attenuation in optically dense sprays, in particular for use in combustion engine research. Images of the fuel sprays are produced by planar laser imaging, where Mie scattered light from a cross section of the spray is imaged onto a CCD camera. The compensation scheme is based on the Beer-Lambert law, which is used here to sum up the loss of light along the path of the laser in the image, and to compensate iteratively, pixel by pixel, for this loss.

Single-cell volume estimation by three-dimensional wide-field microscopy applied to astroglial primary cultures.

Astrocytes, which constitute a prominent part of the number and volume of brain cells, have a high capacity for controlling their volume, and astrocytic swelling is associated with a number of pathological states affecting the CNS. In order to understand the mechanisms for regulating cell volume in astrocytes better, it is of utmost importance to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. For this purpose, a new method was developed to quantify changes in cell volume at the single-cell level. This method is based on three-dimensional (3D) fluorescence imaging obtained by optical sectioning. An automated image acquisition system was developed for the collection of two-dimensional (2D) microscopic images. A deblurring algorithm was implemented in order to restore the originally unfocused image content. Advanced image analysis techniques were applied for accurate and automated determination of cell volume. The sensitivity and reproducibility of the method was evaluated by using fluorescent beads. The techniques were applied to fura-2-labeled astroglial cells in primary culture exposed to hypo- or hyperosmotic stress. The results show that this method is valuable for determining volume changes in cells or parts thereof.

Assessment of facial features with a range camera.

We describe a new method for the quantitative analysis of facial features using a range camera with a three-dimensional measuring program. The range camera consists of a video camera, a light projector, and a computer. The video camera captures a number of images of different light patterns projected on to the face. From these, a "range image" is computed. One fully illuminated intensity image of the face is also taken. By defining anatomical landmarks manually on the intensity image, three-dimensional measurements are automatically obtained from corresponding positions in the range image. The method is highly accurate and precise, and is a simple and reliable way of assessing facial features. We recommend that the system be adapted in clinical practice--for example, in diagnosing syndromes and planning plastic and reconstructive surgery.

A new automated computerized analyzing system simplifies readings and reduces the variability in ultrasound measurement of intima-media thickness.

BACKGROUND AND PURPOSE: A computerized analyzing system with manual tracing of echo interfaces for measurement of intima-media thickness and lumen diameter in carotid and femoral arteries was previously developed by our research group and has been used for many years in several laboratories. However, manual measurements are not only time consuming, but the results from these readings are also dependent on training and subjective judgement. A further problem is the observed drift in measurements over time. A new computerized technique for automatic detection of echo interfaces was therefore developed. The aim of this study was to evaluate the new automated computerized analyzing system. METHODS: The new system is based on dynamic programming and includes optional interactive modification by the human operator. Local measurements of vessel echo intensity, intensity gradient, and boundary continuity are extracted by image analysis techniques and included as weighed terms in a cost function. The dynamic programming procedure is used for determining the optimal location of the vessel interfaces in a way that the cost function is minimized. RESULTS: With the new automated computerized analyzing system the measurement results were less dependent on the reader's experience, and the variability between readers was less compared with the old manual analyzing system. The measurements were also less time consuming. CONCLUSIONS: The new automated analyzing system will not only greatly increase the speed of measurements but also reduce the variability between readers. It should also reduce the variability between different laboratories if the same analyzing program is used. Furthermore, the new system will probably prevent the problem with drift in measurements over time.

Effects of halothane and isoflurane on left ventricular diastolic function during surgical stress in patients with coronary artery disease.

BACKGROUND: The effects of inhalation anesthetics on left ventricular (LV) systolic function are well documented, while the effects of these agents on LV diastolic function have mainly been evaluated in animal studies, with conflicting results. METHODS: We investigated the effects of halothane and isoflurane, when used to control the stress response to sternotomy in 33 patients with coronary artery disease (CAD). LV early diastolic relaxation and end-diastolic stiffness were evaluated from mitral Doppler flow profiles, transesophageal two-dimensional echocardiography, and central hemodynamic measurements. Measurements were performed a) after induction of anesthesia, b) after volume loading, c) prior to surgery and d) during surgery, 10 min after introduction of the inhalation anesthetic. The effects of the anesthetics on Doppler indices reflecting early diastolic relaxation, and on the left ventricular end-diastolic pressure-area (LVED P/A) relationship, were studied. RESULTS: When data obtained during surgical stress were compared to the control situation, we found an increase in the LV filling pressures in both groups, while only the isoflurane group showed an increase in heart rate. An increase in end-systolic LV area and decreased fractional area change was present in the halothane group, while an increase in LV end-diastolic area, and similar changes in the mitral Doppler indices (decreases of deceleration rate and time of early diastolic filling), indicating an impairment of early diastolic relaxation, was present in both groups. Isoflurane induced a displacement of the LVED P/A relationship leftwards from the baseline LVED P/A curve. CONCLUSION: Both halothane and isoflurane impair early diastolic relaxation in patients with CAD, when used to control intraoperative surgical stress. In contrast to halothane, isoflurane induced a change in the LVED P/A relationship, suggestive of an increased LVED stiffness.

Image quality in digital chromosome analysis systems.

This paper reports on an investigation into the differences in image quality of different components used in a digital image processing system for chromosome analysis. As chromosome aberrations are important tools in the cloning of genes, it is important to know if the introduction of computerized analysis systems increases the risk of missing small aberrations. In this investigation the number of visible bands on a number of chromosomes has been used as a measure of quality. The images compared are microscope ocular images, photographs from a microscope built-in camera, digital images from a high and from a standard resolution camera, presented both on screen and print-out on paper. The main conclusions are that: (1) the view in the microscope ocular gives the best resolution, (2) there are risks of losing vital information using the digital image processing system for chromosome analysis, and (3) this risk is significantly reduced when using a high resolution camera.

The effects of nitrous oxide on left ventricular systolic and diastolic performance before and after cardiopulmonary bypass: evaluation by computer-assisted two-dimensional and Doppler echocardiography in patients undergoing coronary artery surgery.

We investigated the effects of nitrous oxide (N2O) on central hemodynamics and left ventricular systolic and diastolic function in 25 patients undergoing coronary artery bypass surgery. All patients were receiving beta-blockers and had good left ventricular function. Global and regional systolic left ventricular performance and diastolic function were determined by computer-assisted analysis of transesophageal echocardiographic (TEE) images, and mitral Doppler flow profiles, respectively. The patients were anesthetized with fentanyl and ventilated with oxygen in air. Hemodynamic and TEE measurements were obtained before and after the introduction of N2O in oxygen (60%/40%), before and after cardiopulmonary bypass (CPB). N2O reduced mean arterial pressure, heart rate, stroke volume, and cardiac output both before and after CPB. Left ventricular global area ejection fraction (GAEF) was not changed by the introduction of N2O, either before or after CPB. N2O induced a significant change in regional wall motion after, but not before CPB, as assessed by the relationship between segmental area ejection fraction (SAEF) and GAEF. Analysis of the mitral flow profile indicated an increase in early diastolic relaxation in the pre-CPB period after introduction of N2O, that was absent in the post-CPB period. We conclude that N2O induces regional wall motion abnormalities and possibly diastolic dysfunction post-CPB.

Morphometry of the optic nerve and retinal vessels in children by computer-assisted image analysis of fundus photographs.

BACKGROUND: The retinal fundus in childhood has a different morphology than in adulthood. Existing methods are not suitable for evaluation of fundus photographs from children. Therefore, a new method for quantitative analysis of fundus morphology utilizing a personal computer-assisted digital mapping system was developed. METHODS: A CCD flatbed scanner is used to digitize fundus photographs, producing computer images which are analyzed on an IBM/AT computer. Area measurements of the optic disc, excavation and peripapillary crescent are made, as well as determinations of the length, branching, tortuosity and distribution of the retinal vessels on the fundus surface. RESULTS: Determination of the inter- and intra-observer variability of the computer-assisted image analysis technique demonstrated good reproducibility. The method is demonstrated using fundus photographs of six normal children and six children with the fetal alcohol syndrome. Typical variations in appearance of optic disc and retinal vessels are seen. CONCLUSION: The system is unique in measuring both the optic nerve head and the retinal vessels and is therefore especially useful for detailed studies of normal and abnormal development of these structures in children.

Automated correction of linear deformation due to sectioning in serial micrographs.

This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi-thin and ultra-thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi-linear, i.e. two first-order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two-dimensional sections and is primarily aimed at three-dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.

Use of computer-assisted image analysis for noninvasive evaluation of oral lichenoid reactions and oral leukoplakia.

The objective of the present study was to elaborate and to evaluate a method in which the clinical appearance of a mucosal lesion could be expressed by means of color and morphologic features extracted from digitized images. The ability to express mucosal changes with image features was evaluated, as was the accuracy of a classification of the lesions on the basis of image feature analysis. Computer-assisted image analysis was performed on digitized color slides from 76 patients with lichenoid reactions that included both oral lichen planus and contact lesions and 20 patients with homogenous oral leukoplakia. Color features were measured according to the intensity-hue-saturation color system, and morphologic features were estimated by tracing the hyperkeratotic area. Hyperkeratinizing and inflammatory tissue reactions could be demonstrated and defined by image analysis. Image features indicating inflammatory tissue reactions were more pronounced in cases of oral lichenoid reactions. Morphologic parameters could express the anatomic appearance of the lesions. The comparison between histopathologic diagnosis and a classification through computerized analysis demonstrated that 90.5% of oral lichenoid reactions could be separated from homogenous oral leukoplakia by linear discriminant analysis.

Effects of adenosine infusion on systolic and diastolic left ventricular function after coronary artery bypass surgery: evaluation by computer-assisted quantitative 2-D and Doppler echocardiography.

The effects of adenosine on central hemodynamics, ST-segment changes, and left ventricular (LV) systolic and diastolic function, determined by transesophageal 2-D and Doppler echocardiography, were investigated in 20 patients shortly after coronary surgery. After control measurements, adenosine was infused at incremental infusion rates (30, 60, and 120 micrograms.kg-1.min-1). Adenosine caused dose-dependent increases in heart rate (68.0 +/- 11.2-74.0 +/- 15.7 bpm), cardiac output (3.23 +/- 0.76-4.17 +/- 0.67 L/min), and stroke volume (48.8 +/- 12.5-56.7 L/min), and stroke volume (48.8 +/- 12.5-56.7 mL), decreases in arterial pressure (84.8 +/- 16.6-63.3 +/- 15.2 mm Hg), and systemic and pulmonary vascular resistances (1994 +/- 510-1106 +/- 309 and 209 +/- 54-116 +/- 58 dyne.s.cm-5, respectively), but no changes in cardiac filling pressures. The mean ST segment was slightly but significantly depressed by adenosine (from 0.003 to 0.019 mV). Analysis of LV wall motion showed that adenosine caused no changes in the global area ejection fraction (GAEF), the segmental area ejection fraction (SAEF), or in the SAEF/GAEF ratio, indicating that no regional wall motion abnormalities appeared. Maximum early and late diastolic flow rates (Emax, Amax), determined by mitral Doppler analysis, increased (from 30.1 +/- 14.8 to 40.1 +/- 24.1 and from 37.8 +/- 15.7 to 46.4 +/- 31.3 cm/s, respectively), as did the deceleration slope of the early diastolic filling (from -151 +/- 67 to -210 +/- 107 cm/s-2), whereas no changes were found in the ratio between Emax and Amax, the deceleration time of early diastolic filling, or the velocity time integrals of early or late diastolic filling.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer analysis in oral lichenoid reactions.

To improve diagnostic procedures and facilitate clinical decision-making, computer-assisted image analysis was performed on color slides from 30 patients with histopathologically verified oral lichenoid reactions. Areas from white hyperkeratotic and adjacent red inflamed areas of the lesions were selected and subjected to image analysis. The digitization of the color slides was done by means of an image scanner, and the digital information was transmitted to a personal computer for subsequent feature extraction and analysis. The different oral lesions were characterized as the difference in mean values between white hyperkeratotic and red inflamed areas, respectively, compared with clinically normal tissue. Statistical analyses were made on three different color systems: Red-Green-Blue (RGB), normalized red-green-blue (rgb), and Intensity-Hue-Saturation (IHS). The results showed statistically significant differences in all color systems for both the hyperkeratotic areas and adjacent inflammatory reactions. A linear correlation was obtained when the results of the image analysis of color variations were compared with a clinical score system for hyperkeratosis and inflammation evaluated by two investigators independently.

Model based dynamic 3D reconstruction and display of the left ventricle from 2D cross-sectional echocardiograms.

In this paper we describe a technique for dynamic three-dimensional (3D) reconstruction of the left ventricle, using boundaries from multiple two-dimensional (2D) echocardiographic views. We use a geometric model of the left ventricle and anatomical landmarks to relate the recorded views to positions within the model. The reconstruction is step-wise refined by replacing model data with recorded contour data.

Computer-assisted 3D analysis of cell distributions in the normal and epileptic cerebral cortex: description of a methodology in progress.

This paper describes software routines that (a) visualizes a stack of several thousands of aligned sequential photographic two-dimensional (2D) images stored in an image processing system; (b) creates a data base containing information about objects identified sequentially from the 2D images; (c) transfers the data base to a graphical terminal; (d) reconstructs a three-dimensional (3D) object space; and (e) supports on-line interaction between the image processing system and the graphical terminal. As an application example, the cell content of a prism of motor cerebral cortex of the cat is reconstructed. Preliminary results from reconstructing human epileptic temporal cortex (cortical microdysgenesia) are also reported.