Toward objective selection of representative microscope images

Scientists wishing to communicate the essential characteristics of a pattern (such as an immunofluorescence distribution) currently must make a subjective choice of one or two images to publish. We therefore developed methods for objectively choosing a typical image from a set, with emphasis on images from cell biology. The methods involve calculation of numerical features to describe each image, calculation of similarity between images as a distance in feature space, and ranking of images by distance from the center of the feature distribution. Two types of features were explored, image texture measures and Zernike polynomial moments, and various distance measures were utilized. Criteria for evaluating methods for assigning typicality were proposed and applied to sets of images containing more than one pattern. The results indicate the importance of using distance measures that are insensitive to the presence of outliers. For collections of images of the distributions of a lysosomal protein, a Golgi protein, and nuclear DNA, the images chosen as most typical were in good agreement with the conventional understanding of organelle morphologies. The methods described here have been implemented in a web server (http://murphylab.web.cmu.edu/services/TyplC).     

Characteristics of the osmotically induced membrane rupture

The phenomenon of reciprocating mechanical oscillations of electrofused erythrocytes was used to investigate the mechanical characteristics of ruptures induced in erythrocyte membranes by colloid osmotic pressure. The rupture characteristics follow an exponentially decaying time function. Time constants determined for opening times of ruptures decreased from 5.5 ms at 10 degrees C to 3.8 ms and 2.0 ms at 40 degrees C for the first and the last observable rupture, respectively. Evidence is given that the diameter of the membrane rupture exceeds the size of a haemoglobin molecule. With repetitive membrane rupturing, the ability of the membrane bilayer and associated structures to heal decreases, owing to the reduced ability to withstand pressure gradients. This change allows oscillating doublets to be classified according to one of three groups: group A showing no development in response to swell times, group B showing a continuous decrease in response to swell times, and group C showing a spontaneous decrease in response to swell times. These results suggest that oscillations cease as a result of defects of membrane healing. Calculations of respective temperature ranges are in agreement with temperature ranges for spectrin denaturation. Thus, conclusions obtained from this study suggest that the spectrin network plays a key role in membrane healing processes after mechanical membrane rupture.     

The role of human red blood cell membrane skeletal proteins in repetitive membrane strain and rupture

The influence of the membrane skeleton on cell membrane deformability, elasticity, and rupture after repetitive cycles of membrane strain, release and rupture was investigated. Human red blood cells were electrofused to doublets, which showed the post-fusion oscillation-movement. Geometrical developments of heat-treated cells were measured and compared to control cells. Alterations of cluster length and fusion zone diameter during repetitive colloidosmotic swelling period grow with heat treatment, and the number of precedent swell phases has minor influence on these values. Irrespective of the treatment, the geometrical doublet configuration at which a membrane rupture is initiated has an almost constant roundness index of 0.89. Increasing heat treatment temperature was shown to affect both deformability and elasticity of the membrane, such that doublets start each swell phase of the oscillation cycle from decreased roundness values. Evidence is given that there is a difference in the mechanical properties between the membrane at the fusion zone and the membrane of native red blood cells.     

The role of human red blood cell membrane skeletal proteins in repetitive membrane strain and rupture

The influence of the membrane skeleton on cell membrane deformability, elasticity, and rupture after repetitive cycles of membrane strain, release and rupture was investigated. Human red blood cells were electrofused to doublets, which showed the post-fusion oscillation-movement. Geometrical developments of heat-treated cells were measured and compared to control cells. Alterations of cluster length and fusion zone diameter during repetitive colloidosmotic swelling period grow with heat treatment, and the number of precedent swell phases has minor influence on these values. Irrespective of the treatment, the geometrical doublet configuration at which a membrane rupture is initiated has an almost constant roundness index of 0.89. Increasing heat treatment temperature was shown to affect both deformability and elasticity of the membrane, such that doublets start each swell phase of the oscillation cycle from decreased roundness values. Evidence is given that there is a difference in the mechanical properties between the membrane at the fusion zone and the membrane of native red blood cells.     

Automated detection of working area of peripheral blood smears using mathematical morphology.

The paper presents a technique to automatically detect the working area of peripheral blood smears stained with May-Grünwuald Giemsa. The optimal area is defined as the well spread part of the smear. This zone starts when the erythrocytes stop overlapping (on the body film side) and finishes when the erythrocytes start losing their clear central zone (on the feather edge side). The approach yields a quick detection of this area in images scanned under low magnifying power (immersion objective x 25 or x 16). The algorithm consists of two stages. First, an image analysis procedure using mathematical morphology is applied for extracting the erythrocytes, the centers of erythrocytes and the erythrocytes with center. Second, the number of connected components from the three kinds of particles is counted and the coefficient of spreading rho(s) and the coefficient of overlapping rho(o) are calculated. The data from fourteen smears illustrate how the technique is used and its performance. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-1/angulo.htm.     

Interactive measurement and characterization of DNA molecules by analysis of AFM images.

BACKGROUND: In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. METHODS: Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. RESULTS: The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. CONCLUSIONS: An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.     

Dynamics of oscillating erythrocyte doublets after electrofusion

Erythrocytes were electrofused with multiple rectangular voltage pulses to show an oscillatory movement, divided into swell phases and pump events. During each swell phase, which lasted from 0.5 s to more than 180 s, the fused cells' (doublets') volume increased by colloid osmotic swelling, and the membrane area was expanded until rupture. Membrane rupture initiated the pump event, where the doublets' volume and membrane area decreased with an almost exponential time course and time constants between 2 ms and 8 ms. Simultaneously, a portion of cytosolic hemoglobin solution was ejected into extracellular space ("jet"). Pump event time constants and swell phase durations decreased with rising chamber temperature, indicating that both parts of the oscillatory movements were determined by physical properties of membrane and liquids. Relative volume change developments express a gradual loss of membrane elasticity during the oscillation, decreasing the elastic forces stored in the membrane. Evidence is given that the first rupture causes a weakening of the membrane at the rupture site. Heat treatment up to 45 degrees C had a negligible effect on swell times, pump time constants, and relative volume changes. A heat treatment of 50 degrees C prevented oscillatory movements. The rupture location accorded with theories of potential induced membrane electropermeabilization.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

Fused Late Endocytic Compartments and Immunostimulatory Capacity of Dendritic–Tumor Cell Hybridomas

Late endocytic compartments, containing MHC class II molecules in antigen presenting cells, fuse to each other in order to deliver antigens to these molecules. We have shown previously that fusion of late endocytic compartments takes place also in hybridomas. Therefore, we investigate here whether the level of fused late endocytic compartments affects the immunostimulatory capacity of hybridomas obtained by the electrofusion of dendritic and tumor cells. The level of fused late endocytic compartments in a single hybridoma cell was assessed and samples of electrofused cells were then cocultured with autologous T cells, resulting in the priming of naïve T cells. To test the immunostimulatory capacity of hybridoma cells, T-cell-induced cytotoxicity of tumor cells was assayed. The results demonstrate that in vitro cytotoxic T cell responses are enhanced if a higher percentage of fused late endocytic compartments is present in the cell population of electrofused hybridoma cells.     

Determination of hemoglobin affinity for oxygen by a polarographic method

The method of determination of hemoglobin affinity for oxygen based on polarographic analysis and the method of half-saturation values P50 calculation are described. A special design of a polarographic cell is proposed for measurements in a wide temperature range. The results of temperature and pH dependences of the hemoglobin affinity for oxygen and erythrocytes of the White-Sea cod are presented.     

Optimal Geometrical Set for Automated Marker Placement to Virtualized Real-Time Facial Emotions

In recent years, real-time face recognition has been a major topic of interest in developing intelligent human-machine interaction systems. Over the past several decades, researchers have proposed different algorithms for facial expression recognition, but there has been little focus on detection in real-time scenarios. The present work proposes a new algorithmic method of automated marker placement used to classify six facial expressions: happiness, sadness, anger, fear, disgust, and surprise. Emotional facial expressions were captured using a webcam, while the proposed algorithm placed a set of eight virtual markers on each subject’s face. Facial feature extraction methods, including marker distance (distance between each marker to the center of the face) and change in marker distance (change in distance between the original and new marker positions), were used to extract three statistical features (mean, variance, and root mean square) from the real-time video sequence. The initial position of each marker was subjected to the optical flow algorithm for marker tracking with each emotional facial expression. Finally, the extracted statistical features were mapped into corresponding emotional facial expressions using two simple non-linear classifiers, K-nearest neighbor and probabilistic neural network. The results indicate that the proposed automated marker placement algorithm effectively placed eight virtual markers on each subject’s face and gave a maximum mean emotion classification rate of 96.94% using the probabilistic neural network.     

Scene-segmentation algorithm development using error measures

Development of scene-segmentation algorithms has generally been an ad hoc process. This paper presents a systematic technique for developing these algorithms using error-measure minimization. If scene segmentation is regarded as a problem of pixel classification whereby each pixel of a scene is assigned to a particular object class, development of a scene-segmentation algorithm becomes primarily a process of feature selection. In this study, four methods of feature selection were used to develop segmentation techniques for cervical cytology images: (1) random selection, (2) manual selection (best features in the subjective judgment of the investigator), (3) eigenvector selection (ranking features according to the largest contribution to each eigenvector of the feature covariance matrix) and (4) selection using the scene-segmentation error measure A2. Four features were selected by each method from a universe of 35 features consisting of gray level, color, texture and special pixel neighborhood features in 40 cervical cytology images . Evaluation of the results was done with a composite of the scene-segmentation error measure A2, which depends on the percentage of scenes with measurable error, the agreement of pixel class proportions, the agreement of number of objects for each pixel class and the distance of each misclassified pixel to the nearest pixel of the misclassified class. Results indicate that random and eigenvector feature selection were the poorest methods, manual feature selection somewhat better and error-measure feature selection best. The error-measure feature selection method provides a useful, systematic method of developing and evaluating scene-segmentation algorithms.     

Mapping the entry of reactive oxygen metabolites into target erythrocytes during neutrophil-mediated antibody-dependent cellular cytotoxicity.

Transmitted Soret band optical microscopy has been used to image the entry and passage of reactive oxygen metabolites across target erythrocytes. Due to the rapid cytosolic diffusion of hemoglobin in comparison to video rates, it was necessary to use erythrocytes with relatively immobilized hemoglobin. To achieve this, erythrocytes from patients with sickle cell anemia were used. The movement of reactive oxygen metabolites across rabbit IgG-opsonized sickle cells was observed in real time. These observations indicate that reactive oxygen metabolites can enter and cross targets in an asymmetric fashion.     

Polymerization in erythrocytes containing S and non-S hemoglobins

We analyzed the effects of protein and water nonideality and of erythrocyte heterogeneity on the polymerization of hemoglobin S in cells where there were significant amounts of non-S hemoglobins, sickle trait (AS), and SC disease. For AS erythrocytes, the calculated predicted results were in good agreement with measured polymer formation as previously reported (Noguchi C.T., D.A. Torchia, and A.N. Schnechter, 1981, J. Biol. Chem. 256:4168-4171). Throughout much of the physiologically relevant oxygen saturation region, polymer was not formed in AS erythrocytes. Measurements of polymer formation in SC erythrocytes as a function of oxygen saturation using 13C NMR are reported here and also are in good agreement with the calculated predicted results. As in sickle (SS) erythrocytes, polymer can be detected in SC erythrocytes in the region above 60% oxygen saturation. The increased polymer formation in SC erythrocytes as compared with AS erythrocytes can be explained in terms of hemoglobin composition and concentration in SC erythrocytes, with the concomitant increase in the proportion of dense cells. These findings provide a basis for understanding the pathophysiology of sickle cell and of SC disease, in contrast to benign sickle trait, in terms of intracellular polymer formation.     

Chromatin texture in high grade prostatic intraepithelial neoplasia and early invasive carcinoma

OBJECTIVE: To evaluate individual nuclei from high grade prostatic intraepithelial neoplasia (PIN) lesions with early invasive carcinoma foci in the area of microinvasion and in the gland in which the microinvasion originated. STUDY DESIGN: High-resolution, digitized images of nuclei from defined locations were recorded and segmented, and karyometric variables were computed. These included a set of 93 features, which form a nuclear signature characterizing the spatial and statistical distribution of the nuclear chromatin. Nuclei in the glandular epithelium were recorded sequentially, along the basal cell layer, at increasing distances from the point of microinvasion and by random selection in the region of microinvasion. RESULTS: At a distance > 60 nuclear locations from the point of microinvasion, the nuclear signatures corresponded to those seen in high grade PIN. Between 40 and 20 nuclear locations removed from the microinvasion focus the signatures began to change gradually until at a distance of 15-5 locations they strongly resembled the signatures seen in adenocarcinoma. The total optical density decreased to values seen in adenocarcinoma, and the nuclear chromatin had finer granularity. While nuclei in high grade PIN followed a widely dispersed total optical density distribution suggestive of wide-ranging aneuploidy, the nuclei in the region of microinvasion exhibited a less dispersed and bimodal total optical density distribution. CONCLUSION: The chromatin texture signatures showed a clear trend: there was an obvious attenuation as the measured nuclei approached the microinvasion area. The decrease in total optical density at the microinvasion might suggest the emergence of one or two clones that can be responsible for the invasive phenotype.     

基于多孔小波融合的PET/CT 图像增强及其评价

目的:为解决融合图像视觉效果增强与量化信息损失之间的矛盾,本文提出一种基于非降采样的多孔小波(àtrous wavelet)分解的PET/CT图像融合方法,使得融合图像既有利于肿瘤诊断又能用于放疗靶区勾画和放射性定量分析。方法:对PET和CT图像分别进行多孔小波分解,以包含肿瘤目标的适当大小的感兴趣区域的清晰度为目标函数,采用Nelder-Mead算法对PET和CT图像高频分解系数之比进行优化获得最终的融合系数,使融合图像充分增加解剖学信息的同时又尽量保持PET图像原有的局部和整体灰度信息。结果:融合图像质量评价表明,本文方法能将有价值的PET功能信息与精确的CT解剖信息结合在一起,并克服传统小波融合损失图像量化信息的不足。结论:基于多孔小波融合的PET/CT图像既能用于肿瘤诊断,又能同时用于肿瘤学放射性计算和适形放疗计划制定等量化研究。     

Generalized ordering parameter S2 for N-H peptide bonding as measure of the conformational flexibility of proteins: comparison of algorithms of S2 calculation from molecular dynamics simulation data

NMR spectroscopy in combination with molecular dynamics (MD) simulation technique is a powerful tool for investigation of biomolecule conformations. In the present study we compare the two algorithms of calculation of the generalized order parameter S2 for the peptide N-H bond from MD simulation data. The program based on this algorithm has been written and the effect of an adjustible parameter on results of calculation was studied. HIV-1 protease MD simulation was performed during 7.36 ns using GROMACS 3.1.4. software package. The calculated order parameter values are in good agreement with those obtained by other authors using NMR.     

Potential Measurement Errors Due to Image Enlargement in Optical Coherence Tomography Imaging

The effect of interpolation and super-resolution (SR) algorithms on quantitative and qualitative assessments of enlarged optical coherence tomography (OCT) images was investigated in this report. Spectral-domain OCT images from 30 eyes in 30 consecutive patients with diabetic macular edema (DME) and 20 healthy eyes in 20 consecutive volunteers were analyzed. Original image (OR) resolution was reduced by a factor of four. Images were then magnified by a factor of four with and without application of one of the following algorithms: bilinear (BL), bicubic (BC), Lanczos3 (LA), and SR. Differences in peak signal-to-noise ratio (PSNR), retinal nerve fiber layer (RNFL) thickness, photoreceptor layer status, and parallelism (reflects the complexity of photoreceptor layer alterations) were analyzed in each image type. The order of PSNRs from highest to lowest was SR > LA > BC > BL > non-processed enlarged images (NONE). The PSNR was statistically different in all groups. The NONE, BC, and LA images resulted in significantly thicker RNFL measurements than the OR image. In eyes with DME, the photoreceptor layer, which was hardly identifiable in NONE images, became detectable with algorithm application. However, OCT photoreceptor parameters were still assessed as more undetectable than in OR images. Parallelism was not statistically different in OR and NONE images, but other image groups had significantly higher parallelism than OR images. Our results indicated that interpolation and SR algorithms increased OCT image resolution. However, qualitative and quantitative assessments were influenced by algorithm use. Additionally, each algorithm affected the assessments differently.     

Differential time‐dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum‐infected hemoglobinopathic erythrocytes

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid‐osmotic model on the grounds of time‐resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid‐osmotic model can predict time‐dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model‐predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid‐osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum‐infected erythrocytes. The contribution of vesiculation to the malaria‐protective function of hemoglobin S is discussed.     

STUDIES IN HISTOCHEMISTRY : LVII. Determination of the Total Dry Mass of Human Erythrocytes by Interference Microscopy and X-ray Microradiography

The total dry mass of human erythrocytes was determined by both interference microscopy and x-ray microradiography. The determination of mass per unit area, and calculation of total dry mass per cell were simplified by changing the shape of the cells to spheres which were then flattened to discs of constant thickness when smeared on glass slides for measurement of fixed cells by interferometry, and to oblate spheroids when smeared on parlodion-coated slides for measurement of fixed cells by x-ray absorption. From x-ray measurements of 100 smeared and alcohol-fixed cells a mean dry mass per cell of 33.7 x 10^-12 g was obtained. Interference measurements of 100 fresh cells suspended in isotonic saline gave a mean value of 32.4 x 10^-12 g while interference measurement of 100 smeared and alcohol-fixed cells gave a mean value of 30.8 x 10^-12 g. The first two values compare well with a mean corpuscular hemoglobin of 31.2 x 10^-12 g, obtained from determinations of erythrocyte count and hemoglobin, since 95 per cent of the dry mass of the cell is hemoglobin. The difference in interference values between the fixed and fresh cells is possibly due to a difference between the specific refractive increment of alcohol-denatured hemoglobin and that of the unmodified substance. The value for the latter was used since that of the former is unknown.