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Interactive image analysis system to determine the motility and velocity of cyanobacterial filaments.

An interactive image analysis system has been developed to analyse and quantify the percentage of motile filaments and the individual linear velocities of organisms. The technique is based on the "difference" image between two digitized images taken from a time-lapse video recording 80 s apart which is overlaid on the first image. The bright lines in the difference image represent the paths along which the filaments have moved and are measured using a crosshair cursor controlled by the mouse. Even short exposure to solar ultraviolet radiation strongly impairs the motility of the gliding cyanobacterium Phormidium uncinatum, while its velocity is not likewise affected. These effects are not due to either type I (free radical formation) or type II (singlet oxygen production) photodynamic reactions, since specific quenchers and scavengers, indicative of these reactions, failed to be effective.

Cell Movement↗

Quantitation of microvessel density in squamous cell carcinoma of the head and neck by computer-aided image analysis.

OBJECTIVE: Angiogenesis is the formation of new blood vessels from a preexisting vascular network. In healthy individuals it is normally suppressed and observed only transiently during development, reproduction and wound healing. However, growth, invasion, and metastasis of most solid tumors are dependent on angiogenesis. Without formation of new blood vessels, also termed as neovascularization, tumors cannot exceed a size of about 1 mm3. Therefore, neovascularization is a basic requirement for nutrition and oxygenation of tumor cells. Numerous studies in different solid as well as non-solid tumors have evaluated the prognostic value of tumor neovascularization. In solid tumors the increased microvessel density, the pathological correlate to tumor neovascularization, has been linked to a worse prognosis of the disease. The aim of the current study was to assess the prognostic value of tumor neovascularization for recurrences in squamous cell carcinoma of the head and neck by determining microvessel density. Data was collected using an automated-computerized method and as well as a manual counting method. BASIC RESEARCH DESIGN: Immunohistochemistry was performed to detect intratumoral microvessels in tumor samples of 50 patients with squamous cell carcinoma of the head and neck. We used a monoclonal mouse antibody directed against the CD34 antigen. After immunostaining, the entire tumor section was scanned microscopically at low power (x 40) to identify hot spots, which are the areas of highest neovascularization. Individual tumor microvessels were then counted under high power (x 200) to obtain a vessel count in a defined area, and the average vessel count in 4 hot spots was taken as the microvessel density. Microvessel counting was performed twice by computerized method, as well as manually by two independent investigators without any previous knowledge of patients' pertinent clinical data. Subsequently, both counting techniques were statistically compared with each other. RESULTS: On computer-aided image analysis an increased microvessel density was significantly correlated with recurrence of disease (p = 0.02). Repetitive computer counts yielded similar results (p = 0.08), whereas repeated manual counts by two investigators varied significantly (p = 0.04). However, no further statistical correlations between microvessel density and patients clinical data i.e. tumor status, lymph node status, overall survival, or disease free interval could be found. Furthermore, estimation of overall survival of patients with an increased microvessel density by Kaplan-Meier curves revealed non-significant results. CONCLUSION: There is mounting evidence that suggests, that assessment of tumor neovascularization might provide a novel approach of prognostication in patients with squamous cell carcinomas of the head and neck. In particular, in the present study, the degree of angiogenesis of a tumor, as assessed by microvessel density, was found to be correlated with recurrent disease in squamous cell carcinoma of the head and neck. Computer aided image analysis, an automated technique, constitutes a time-efficient and reproducible technique for quantification of tumor vascularization. We suggest that this computerized microvessel determination could be used as a reliable method for microvessel counts, which, furthermore, seems to be superior to manual counting. However, for a reliable and reproducible assessment of tumor neovascularization, validation procedures and quality control protocols are mandatory.

Adult↗

Image analysis measurements of roughness by texture and fractal analysis correlate with contact profilometry.

Surface properties of a biomaterial are important factors that govern in part its biocompatibility. Among them, surface roughness is now recognized as a very important factor for cell interactions. Surface roughness (Ra) is routinely measured by contact profilometry but other methods are presently usable. We compared two methods (contact profilometry and image analysis of scanning electron microscopic images SEM) on a series of 12 titanium test pieces. The texture analysis of SEM pictures was done by the heterogeneity and run-length methods. Fractal geometry was also used with the "skyscraper" and "blanket" methods providing respectively the D(SKY) and D(BLANK) fractal dimensions. The fractal dimension of the profilometric curve was also computed (D(MINK)). Computer-simulated textures were used to evaluate the pertinence of the algorithms. A significant correlation was found between Ra and all the texture descriptors except heterogeneity. The correlation coefficient was dependent on the microscopic magnification. The fractal dimension of the curve was correlated with D(SKY) and D(BLANK). Run-length, D(SKY) and D(BLANK) were highly correlated, independent of the magnification used, a finding related to the self-similarity of the images. Image texture analysis can be a useful alternative to profilometry with brittle or soft materials or with objects having a complex shape.

Algorithms↗

Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata.

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 Mr oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (alpha 2 beta gamma delta) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the delta chains. In agreement with previous work (Cartaud et al., 1980) the H-form appeared as "doublets" of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 +/- 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublets are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the delta chain within the structure. Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified. Tentative assignment of structural details to individual subunits was deduced from an examination of alpha-bungarotoxin-labeled doublets. The alpha subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).

Animals↗

[Use of automated microscopic image analysis for the determination of microscopic hyphomycetes (Fusarium sp.)].

The method of automated microscopic image analysis was tested in intention to support the taxonomy of Fusarium. The catalogue of morphometric object features of the software system AMBA provides size and shape parameters which can be utilize to discriminate populations of macroconidia of different Fusarium species and varieties. The applied colouring of macroconidia (methylene blue) has no significant effect on these features. The results of statistical analysis show fitness of chosen technique.

Fusarium↗

Desktop image analysis: workstations of the future.

We report the use of an interactive graphics-oriented workstation to explore quantitative information extracted from medical images. At the University of California at Los Angeles, a Macintosh II personal computer operates as a stand-alone system and images are imported from a central PACS (picture archiving and communication system) server through an Ethernet network. The image-analysis program contains a complete set of general-purpose tools for image manipulation and processing, as well as specific tools for clinical analysis of cardiac and vascular images obtained by various techniques. Most of these features were designed to communicate information through color-coded graphic displays. Ten novice users were given a brief introduction to the program and asked to perform eight analytic tasks. The overall success rate was 95%, and the subjects considered the program easy to use without a manual or formal training. We conclude that with a well-designed graphic interface, physicians without training in the use of computers can easily learn to manipulate and analyze medical images.

Computer Graphics↗

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold.

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

Cyanobacteria↗

[A quantitative study on DNA content of hepatocellular carcinoma by image analysis technique].

DNA content was quantitatively determined in 38 hepatocellular carcinoma (HCC) by image analysis technique using paraffin-embedded sections stained with Feulgen method. The results showed that diploid/near diploid DNA content (D/ND) and aneuploid DNA content (AN) were found in 15.8% and 84.2% of HCC. The incidences of tumor capsule invasion and cancerous thrombus formation were 84.4% and 87.5% in AN HCC which are significantly higher than in D/ND HCC (33.3% and 50%) (P less than 0.05). The tumor size of HCC was related to DNA content, 66.7% of D/ND HCC was less than or equal to 3 cm in diameter, whereas 75% of AN HCC was greater than 3 cm across (P less than 0.05), suggesting that HCC at 3 cm in size may be a critical point at which the biologic characteristics of HCC would change. There was no correlation between the DNA content and pathologic grading of HCC. It is suggested that the development of AN stem line with a broad distribution of DNA content be an important biologic marker of HCC with increased malignancy and the quantitative analysis of DNA content be able to provide an objective and quantitative reference for evaluating the biologic characteristics. The above observation may be taken as a valuable supplement to the pathologic grading of HCC.

Adult↗

[Quantitative basis for medical imaging analysis: Information theory and Bayesian inference].

One of the main issues in radiology is image analysts and report. The two stages of this process are the extraction of the information from the image and the diagnostic exercise. This paper is a theoretical analysis of image interpretation from a quantitative perspective, using information theory and Bayesian inference, and relating it to the concepts of evidence based medicine. It is concluded that the amount of information is dependent on the previous state of knowledge and the acquaintance with the clinical history and complementary exams. The three determined stages of the diagnostic process are a priori, conditioned by the image and a posteriori analyses. The first stage is related to the degree of information and knowledge of the case. The second stage is dependent on the conditional probability for the presence of image signs of the disease and it is thus directly dependent on the sensitivity and specificity of the exam. The third stage is dependent of the previous stages, that in the simplest case can be expressed in function of the odds or likelihood ratios. These three stages allow to reach a decision about the usefulness of a given exam and they orient the questions that a given image may answer. They also orient the research on image analysis for an adequate decision making, minimizing diagnostic uncertainty.

Bayes Theorem↗

[Image analysis of cell nuclear morphology and DNA content in osteosarcoma of the jaws].

The measurement of nuclear morphology and quantitative analysis of nuclear DNA content were made by Interactive Image Analysis System in 47 osteosarcomas of the jaws. The results showed that with a decreased degree of differentiation, the cell nuclei of osteoblastic and chondroblastic osteosarcomas came to be large and irregular, those of fibroblastic osteosarcomas, however, to be small and round. It was revealed that high-grade osteosarcomas had increased amounts of DNA and that the mean of DNA content, proportion of hyperploid cells and tumor ploidy were found to be of important prognostic value.

Cell Nucleus↗

Decreases in blood perfusion of the anterior cingulate gyri in Anorexia Nervosa Restricters assessed by SPECT image analysis.

BACKGROUND: It is possible that psychopathological differences exist between the restricting and bulimic forms of anorexia nervosa. We investigated localized differences of brain blood flow of anorexia nervosa patients using SPECT image analysis with statistic parametric mapping (SPM) in an attempt to link brain blood flow patterns to neurophysiologic characteristics. METHODS: The subjects enrolled in this study included the following three groups: pure restrictor anorexics (AN-R), anorexic bulimics (AN-BP), and healthy volunteers (HV). All images were transformed into the standard anatomical space of the stereotactic brain atlas, then smoothed. After statistical analysis of each brain image, the relationships among images were evaluated. RESULTS: SPM analysis of the SPECT images revealed that the blood flow of frontal area mainly containing bilateral anterior cingulate gyri (ACC) was significantly decreased in the AN-R group compared to the AN-BP and HV groups. CONCLUSIONS: These findings suggest that some localized functions of the ACCare possibly relevant to the psychopathological aspects of AN-R.

Anorexia Nervosa↗

CT image analysis of the vertebral trabecular network in vivo.

A method of computed tomography (CT) image analysis of lumbar vertebrae has been developed, providing a visualization of the trabecular network as it is represented in a 1.5 mm-thick CT image. We measured the length of the network and the number of discontinuities found in the image. The ratio of these measurements was called the "trabecular fragmentation index" (TFI). CT images from 71 women between the ages of 50 and 59, and 94 women between the ages of 60 and 69 were divided into three groups according to quantitative computed tomography (QCT) vertebral density and to the presence or absence of crushing and fractures. The measure of the network length versus the vertebral area was significantly higher in normal subjects than in osteoporotics. A TFI threshold at 0.195 could separate the normal subjects, regardless of the decade, from osteoporotic ones. In females between 50 and 69 years of age, TFI was 0.166 (SD = 0.031) for the normal group and 0.248 (SD = 0.082) for osteoporotics. The osteopenic group without fractures but low bone mineral density (BMD) showed an intermediate TFI of 0.195 (SD = 0.05), placing this population on both sides of the threshold. Correlation between TFI and BMD was only -0.60. TFI could provide new information in vivo about the state of trabecular structure, particularly in the osteopenic group.

Aged↗

CellProfiler: image analysis software for identifying and quantifying cell phenotypes.

Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining).

Dose-Response Relationship, Drug↗

The determination of growth rates of individual colonies in agarose using high-resolution automated image analysis.

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation-modifying agents.

Adenocarcinoma↗

[Image analysis of tumor cells from common intracranial neoplasms on frozen and paraffined slices].

Image analysis technique was used to measure four parameters [the form factor (FF), area, perimeter and diameter] of nuclei in 76 cases of common intracranial tumors on forzen and paraffined slices. The results showed that the area, perimeter, diameter of nuclei on frozen slices were much larger than those on paraffined slices while the FF showed no significant difference between two kinds of slices (P > 0.05). The parameters of the area, perimeter, and diameter of nuclei on two kinds of slices became larger and the parameters of FF became smaller as the grade of astrocytoma ascended. It is considered that the results of present study will be helpful to the diagnosis of common intracranial tumors on frozen slices.

Astrocytoma↗

Image analysis of the distal radius trabecular network using computed tomography.

Bone texture analysis might provide information about bone structure in a noninvasive manner. In a prospective case-control cross-sectional study we investigated the value of computed tomography (CT) image analysis of the distal radius in the assessment of osteoporosis. Twenty patients suffering from postmenopausal osteoporosis were studied and compared with 21 age-matched controls. Eight slices were selected in each patient: four consecutive coronal slices and four consecutive cross-sectional slices. Bone texture analysis was performed using statistical, fractal and structural methods leading to the measurement of 32 features. Structural variables derived from histomorphometric parameters were measured after segmentation from a binary or a skeletonized image. Bone mineral density was measured by dual-energy X-ray absorptiometry both at the lumbar spine and the femoral neck. Eight of the 9 statistical features were significantly different in osteoporotic women as compared with controls (coronal slices, p < 0. 05). Seven structural variables were statistically different between the two groups on coronal slices (p < 0.05): valley surface area, bone volume/tissue volume, trabecular partition, Euler's number, trabecular bone pattern factor, node-to-node strut count and terminus-to-terminus strut count. The most significant results on coronal slices (p < 0.01) concerned 4 structural features: trabecular partition, Euler's number, trabecular bone pattern factor and terminus-to-terminus strut count. Three features were statistically different (p < 0.01) between the two groups on cross-sectional slices (skeletonization from gray levels). A few features yielded by texture analysis were correlated with both lumbar spine and femoral neck bone mineral density, but the level of these correlations was weak (r < 0.5). In conclusion, CT image analysis of the distal radius is a useful tool for characterizing bone texture alterations in osteoporotic women. These findings are in keeping with microarchitectural osteoporosis-related changes diagnosed on bone biopsies.

Aged↗

Measurements of lens transparency or its disturbances by densitometric image analysis of Scheimpflug photographs.

Scheimpflug photography of the anterior eye segment with densitometric image analysis to measure lens transparency has proven its value in long-term follow-up studies of lens changes. The necessary technical conditions of the SL 45 Topcon camera guarantee high reproducibility. The technical control with respect to quality in case of repeated photography is ensured by an internal standard, the camera being equipped with a five-step scale of known density values. External standardization is done by taking the densitometer height of the cornea as standard. The coefficients of variation of internal and external standards were nearly equal (approximately 5%) over the 9-month period of observation of 100 patients (3,200 photos). The coefficients of variation correspond to the standard error found with repeated lens photos of volunteers' eyes evaluated by analysis of variance of the respective data sets. The standard error for distance measurements on the x-axis (cornea thickness, depth of anterior chamber, lens thickness, etc.) is less than 3%. Evaluation of follow-up examinations may most successfully be done by densitometric planimetry, which, in case of follow-up studies on opacified lenses, is superior to the usual measurements of densitometer height. The light scatter registered by Scheimpflug photography is highly individual. This is demonstrated by densitograms of normal lenses of persons of different age groups. Besides linear densitometry, various other procedures of densitometric planimetry are possible. The best results so far were obtained by multilinear densitometry, but the enormous amount of data obtained in this way renders routine application rather difficult.

Cataract↗