Computer-aided Image Analysis Research Articles

The use of protein synthesis elongation factor EF-Tu as internal calibration standard in two-dimensional electrophoretic studies of differentiation in Streptomyces.

Protein synthesis elongation factor EF-Tu is presented as an internal calibration standard for quantitative analysis of two-dimensional (2-D) protein electrophoresis gels. EF-Tu was selected on the basis of concentration measurements in cell-free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one-dimensional (1-D) and 2-D electrophoresis gels. The results demonstrated that the amount of EF-Tu synthesized in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF-Tu an ideal internal protein standard for quantitation of protein spots on 2-D electrophoresis gels. For such quantitative analysis we developed a computer-aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.

AUTOMATION IN MAMMOGRAPHY: COMPUTER VISION AND HUMAN PERCEPTION

Mammographic screening programmes generate large numbers of highly variable, complex images, most of which are unequivocally normal. When present, abnormalities may be small or subtle. Two processes critical to the success of screening programmes are the perception of potential abnormalities and the subsequent analy-sis of each detected lesion to determine its clinical significance. The consequences of errors are costly, and in many screening centres, films are read by two radiologists in an attempt to reduce errors. The prime objective of our research is to improve the accuracy of the detection and analysis of breast lesions by providing radiologists with computer-aided digital image analysis tools. In this paper we focus on the detection and analysis of mammographic microcalcifications. We describe a philosophy of research aimed at generating useful computer-based aids for radiologists. Firstly, it is necessary to accurately identify specific tasks which are difficult for the human observer. Having correctly identified a problem, appropriate computer vision methods must be developed and their performance evaluated. It is then important to determine effective ways of using such methods to aid radiologists, and it is essential to prove that the effect on radiologists’ performance is entirely beneficial. We present results of experiments to determine factors affecting radiologists’ perception of microcalcifications, and to investigate the effects of attention-cueing on detection performance. Our results show that radiologists’ performance can be significantly improved with the use of prompts generated from automatically-detected microcalcification clusters. We describe a new method for the delineation of mammographic abnormalities based on the analysis of multiple high quality X-ray projections of excised lesions. Biopsy specimens are secured inside a rigid tetrahedron, the edges of which provide a reference frame to which the locations of features can be related. A three-dimensional representation of an abnormality can be formed and rotated to resemble its appearance in the original mammogram.

Automated histomorphometry of interstitial reactions in the heart

A method is presented suitable to measure changes in area densities of the total Sirius red-stained interstitial tissue in longitudinal sections of rat hearts by means of computer-aided image analysis considering at the same time the behavior of six regions and changes in the percentage of thick connective tissue fibers in the total interstitial tissue. Verification tests were directed to the influences of preparation, adjustment of the grey-level thresholds, and identification of the various regions, to marginal conditions of measurement and the investigators. The reliability of the method with acceptable total errors is demonstrated.

Immunohistochemical study of catecholamine enzymes and neuropeptide Y (NPY) in the rostral ventrolateral medulla and bulbospinal projection.

The purpose of this study was to determine whether neuropeptide Y (NPY) terminals in the intermediolateral spinal cord originate from the rostral ventrolateral medulla (RVLM). Immunohistochemical staining of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and NPY in the rat brainstem and spinal cord were performed in this study in order to examine consequences of lesions of the RVLM and of intracisternal injections of 6-hydroxydopamine (6-OHDA) on catecholamine and NPY immunoreactivity in the intermediolateral column (IML) of rats. In addition, ricin, a retrograde neurotoxin, was applied in the superior cervical ganglion (SCG) to determine its effect on catecholamine and NPY immunoreactivity in the IML. Computer-aided image analysis was used to quantify the immunohistochemical changes in the RVLM and spinal cord. The results demonstrated that many catecholamine- and NPY-containing neurons and/or fibers existed in the RVLM and their terminals were found in the IML. After administration of 6-OHDA intracisternally, the catecholamine and NPY immunoreactivities were decreased both in the brainstem and IML of the spinal cord. Following unilateral microinjection of 6-OHDA into the RVLM, the number of NPY- and catecholamine-containing neurons decreased and there was a reduction in neuron terminals on the ipsilateral side. After injection of ricin into the SCG, the catecholamine and NPY neurons of the medulla were not affected, whereas their terminals in the IML decreased ipsilaterally. These results indicate that most of the catecholamine- and NPY-immunoreactive terminals found in the IML originated in the RVLM. These terminals appear to project towards the superior cervical ganglia.

Determining chromatin structure by computer-aided image analysis

In the present research, we address the problem of how chromatin fibers are ordered in band and interband regions along the length of a Drosophila polytene chromosome. Our approach employs image processing as a preliminary step to amplify the image contrast. Then, computerized pattern recognition methods are used to study how the chromatin is arranged.Polytene chromosomes were isolated from salivary glands and squashed on glass microscope slides. The slides were immersed in liquid nitrogen and the coverslips were removed with a razor blade. Small droplets of a polymer developed in our laboratory called HACH (a mixture of 2- hydroxyhexanedial, carbohydrazide and hydrazine) were then placed over individual chromosome spreads and the slides were left at 26°C overnight to allow for HACH polymerization. HACHembedded samples were removed from the slides, mounted on resin blocks, trimmed and thinsectioned at a thickness of approximately 100 nm. Sections were floated onto formvar-coated gold grids and viewed with a Zeiss 10C transmission electron microscope operated at 80kV.

Granulomatous reaction and tissue remodelling in the cutaneous lesion of chromomycosis.

The cell-mediated immune reaction was studied in the cutaneous lesion of chromomycosis, using monoclonal antibodies against polymorphonuclear neutrophils, macrophage and lymphocyte subsets, endothelial and fibroblast cells. In addition, immunostaining of the main degradative enzymes (neutrophil elastase and interstitial collagenase) and certain important cytokines (transforming growth factor-beta, tumour necrosis factor-alpha and interferon-gamma) suggested an explanation for the granulomatous reaction and the associated tissue remodelling. The distribution pattern of neutrophils and macrophage subsets, observed by computer-aided image analysis, suggests that the in situ persistence of fungi is the main pathological factor.

Anatomical Sequence and Morphometric Analysis during Somatic Embryogenesis on Cultured Cotyledon Explants of Camellia japonica L.

This article describes the origin and anatomical development of somatic embryos differentiated on Camellia japonica L. cotyledons cultured on Murashige and Skoog's medium containing 1 mg l-1 of 6-benzylaminopurine. Only the abaxial surface of the cotyledon explants was morphogenetically competent. Embryos developed in abaxial parenchymatic protuberances or nodules arising by dedifferentiation and active cell division in the epidermis and subepidermis. After 12-15 d in culture, successive divisions at the surface of the nodules led to the formation of embryogenic precursor cells which dedifferentiated into embryogenic cells; most somatic embryos apparently had a multicellular origin from multicellular proembryonal complexes, though a number of few-celled proembryos within a thick common wall seemed to have originated unicellularly. Between days 24 and 27, somatic embryos at the heart-shaped, torpedo-shaped and cotyledonary stages were apparent. Computer-aided image analysis of the histological events showed a progressive increase in the nucleus-to-cell area ratio. During the first 7 d culture the explants exhibited a rapid decline in protein body content, which was high in the initial cotyledon, and an increase in starch content. Developing nodules strongly PAS-positive, but starch content subsequently declined in the tissues underlaying embryogenic areas and reached a minimum when somatic embryos developed.

Identification of serous-like cells in the surface epithelium of human bronchioles

The conductive airways of the mammalian lung are lined by several morphologically distinct cell types, both ciliated and non-ciliated. Of the non-ciliated secretory cells, mucous (goblet), Clara and neuroendocrine cells have been identified in adult human bronchiolar epithelium but, thus far, the serous cell has not. We have examined human membranous and respiratory bronchioles from macroscopically normal peripheral lung (n = 5 cases). For ultrastructural studies, a minimum of five nucleated, non-ciliated cell profiles, containing electron-dense secretory granules, were selected at random from both bronchiolar levels in each case, such that a total of 60 cells was examined. Data from the computer-aided image analysis of the cells indicated that two populations existed, differing in both granule area and granule number per cell p < 0.0005 by discriminant analysis. By visual inspection, the cells fell neatly into two groups: those which, were predominantly serous-like in type, and those which were "Clara". In the membranous bronchioles, serous-like cells had a mean(SEM) granule area per cell of 3.67(0.62) microns 2 and Clara cells 0.47(0.07) microns 2 (p < 0.001). Also, in the membranous bronchiole, the mean(SEM) number of granules in serous-like cells was 40(4.7) and in Clara cells 10(1.1) (p < 0.001). At the respiratory bronchiolar level, the corresponding means were similar to those of the membranous bronchioles and, likewise, serous and Clara cells were significantly different. Thus, our data, indicate that serous cells are present in the adult human bronchiole.

Digital dynamic range expansion applied to X-ray densitometric analysis of total hip replacement

Due to the presence of the stiff prosthetic stem fitted in the medullary canal during total his replacement, the surrounding cortex of the femur changes its density over time. This bone remodelling takes place with every type of total hip prosthesis; however, its intensity may vary between prostheses and patients. In the worst cases this process can lead to the late failure of the implant. To monitor such bone density evolution, we are developing a tailored Computer-aided Densitometric Image Analysis system (the major part of this our system uses an 8-bit commercial hardware with 256 levels of grey). The equivalent dynamic range of an X-ray picture is about 10 bits. In this paper we present a method to overcome these hardware limitations by improving the software. Using a double-exposure acquisition it is possible to build a 9-bit image that is good enough for most applications involving bone density measurement.

Comparison between wedge and needle biopsies for evaluating the degree of cirrhosis

To examine whether the biopsy method could affect histological evaluation, the volumetric ratio of human liver parenchyma was estimated in specimens from subcapsular and intralobar areas, and also in specimens obtained by needle biopsy (Tru-Cut needle). A new method of morphometric analysis was performed using a computer-aided color image analyzer. Eighteen cirrhotic, 7 fibrotic, and 4 normal liver biopsies were taken during hepatic resection and analyzed. The parenchymal cell volume ratio in the intralobar area was significantly correlated with that in the subcapsular area, and less significantly with needle biopsy samples (r = 0.844, p < 0.001; r = 0.577, p < 0.01, respectively). Both showed one-to-one correspondence. These results suggest that both wedged and needle biopsy samples are appropriate for assessing the degree of fibrosis or cirrhosis, although the sampling variability of the latter is greater than the former.

Image analysis in the evaluation of biomaterials

An examination regime, based on a computer-aided image analysis system, has been developed for the quantitative evaluation of the local tissue response to biomaterials. This procedure involves the immunoenzymic staining of tissue sections using monoclonal antibodies specific for certain inflammatory cell types. An avidin-biotin-horseradish peroxidase staining method is used to identify antibody binding sites and the sections are assayed using a computer-aided image analysis system. This regime facilitates the rapid and accurate measurement of 30 cell related parameters in sections stained for macrophages, polymorphonuclear leucocytes, and other cells.

Inference systems for automated image analysis.

The radiodiagnostic process is a complicated activity involving the integration of knowledge from low-level image features into more abstract, higher-order entities. This involves spatial and density information at a lower level and area and features at a higher level. The spatial density distribution in a radiographic image is not uniquely related to the three-dimensional structure of the object. Therefore, more information other than just first-order density characteristics of radiographs must be utilized to improve automated interpretation of the image. Prior knowledge of the size, shape and location of anatomical structures and pathognomic features is very useful for improving the process of computer-aided image analysis. Inference systems as used in expert systems can be applied to facilitate the integration of information obtained from the patient and the radiograph in the diagnostic process.

Effects of the pericardium on left ventricular diastolic filling and systolic performance early after cardiac operations

To determine whether closure of the pericardium after cardiac operations affects the filling characteristics and systolic performance of the left ventricle, we measured left ventricular volume, pressure, cardiac index, and stroke work index in 10 patients between 11 and 15 hours after cardiac operations, with the pericardium first closed and then open. At the time of operation, radiopaque tantalum markers were inserted in the left ventricular myocardium to outline the chamber in the 30-degree right anterior oblique projection, and the pericardium was closed by a continuous polypropylene suture exteriorized at both ends of the sternotomy. The patient was then transferred to the surgical intensive care unit, where left atrial pressure was measured via a fluid-filled catheter, left ventricular pressure with a micromanometer-tipped catheter, and myocardial oxygen consumption via a coronary sinus catheter. Left ventricular volume was measured by computer-aided analysis of fluoroscopic images (recorded at 30 frames per second) of the implanted myocardial markers. Left atrial pressure was maintained at target values of 10, 15, and 20 mm Hg by intravenous augmentation of blood volume. Left ventricular and left atrial pressures and volumes were measured with the pericardium closed; the pericardium was then opened by withdrawal of the pericardial suture. Radiopaque clips on the pericardial edges confirmed opening of the pericardium seconds after withdrawal of the suture. Repeated measurements of left ventricular pressures and volumes were then made at the target left atrial pressures with the pericardium open. End-diastolic volume index, peak positive time derivative of pressure, stroke work index, and cardiac index all increased significantly when the pericardium was opened (p < 0.001). Thus we found the following: (1) At physiologic pressures, the pericardium had a significant constraining effect on diastolic filling of the left ventricle, and (2) opening of the pericardium resulted in increased cardiac index and stroke work index. These increases may be attributed to the Frank-Starling response to increased left ventricular preload. The demonstrated improvement in left ventricular systolic performance should be considered when contemplating closure of the pericardium after cardiac operations, especially in patients with preoperative left ventricular dysfunction.

Application of testosterone accelerates oligodendrocyte maturation in brains of zebra finches.

The effects of exogenously applied hormones on glial cell maturation of telencephalic song motor centers, midbrain and cerebellar structures was studied in juvenile male zebra finches. Testosterone was administered and the development of oligodendrocytes was studied using immunochemistry and computer aided image analysis on silver impregnated brain sections. A testosterone induced acceleration of oligodendrocyte maturation could be detected in several brain areas by using the monoclonal antibody O 10 recognizing an oligodendrocyte-specific cell surface antigen and by the silver impregnation for myelin. The increase in myelin density was higher in the testosterone treated animals than in the control animals in the forebrain and in the cerebellum, whereas two regions in the midbrain showed no difference between treated and controls.

Sperm Axoneme in Some Apterygote Insects Examined by Computer-aided Image Analysis

Sperm tail axonemes from three insect species, representing the three apterygote orders Diplura, Archeognatha and Zygentoma, have been examined by high resolution electron microscopy. The samples were fixed in a mixture of glutaraldehyde and tannic acid followed by uranyl acetate post-fixation and the electron micrographs were subjected to computer analysis in order better to characterize the minute details of the axonemal microtubules. The dipluran Campodea was seen to have a row of accessory microtubules composed of 13 protofilaments, the archeognath Machilinus to have two rows of accessory microtubules composed of 16 protofilaments. and the zygentoman Lepismodes to have accessory microtubules in contact with the flagellar doublets and with a wall containing 16 protofilaments and an electron-dense lumen. The axonemal structure in Zygentoma is the one most similar to those of the pterygote insects. The protofilaments of the axonemal doublets were seen to have a somewhat widened interspace between some pairs of protofilaments and these ‘gaps’ coincided with the location of the basal parts of the outer dynein arm, the inner dynein arm, the spoke, and the intertubular material. It hence seems that these microtubule-associated structures influence the architecture of the microtubular wall.

Direct evidence for the intact hepatocyte theory in patients with liver cirrhosis

An attempt was made to compare various morphometric parameters, including total hepatocyte number, with the in vivo clearances of aminopyrine and antipyrine in 26 cirrhotic and 14 noncirrhotic patients to evaluate the intact hepatocyte theory. Morphometric analysis was performed with a newly developed method using a computer-aided color image analyzer. Aminopyrine clearance was significantly correlated with liver volume (r = 0.434; P less than 0.05), parenchymal cell volume (r = 0.574; P less than 0.001), and most strongly with total hepatocyte number (r = 0.614; P less than 0.001) in all patients. Significant correlations were also observed between these three parameters and antipyrine clearance (r = 0.367, P less than 0.05; r = 0.663, P less than 0.001; and r = 0.807, P less than 0.001, respectively). The mean aminopyrine clearance per individual hepatocyte showed no significant difference between cirrhotic and noncirrhotic patients (3.52 +/- 1.60 x 10(-10) mL/min vs. 3.65 +/- 1.50 x 10(-10) mL/min, respectively; P greater than 0.10). Similar results were obtained for antipyrine clearance per hepatocyte (7.35 +/- 2.27 x 10(-11) mL/min for cirrhotics vs. 6.16 +/- 1.07 x 10(-11) mL/min for noncirrhotics; P greater than 0.10). Thus, the intrinsic clearances of drugs per individual hepatocyte, as originally proposed in the intact cell hypothesis, were directly evaluated for the first time, lending strong support to the intact hepatocyte theory. (Gastroenterology 1992 Apr;102(4 Pt 1):1351-5)

Direct evidence for the intact hepatocyte theory in patients with liver cirrhosis

An attempt was made to compare various morphometric parameters, including total hepatocyte number, with the in vivo clearances of aminopyrine and antipyrine in 26 cirrhotic and 14 noncirrhotic patients to evaluate the intact hepatocyte theory. Morphometric analysis was performed with a newly developed method using a computer-aided color image analyzer. Aminopyrine clearance was significantly correlated with liver volume (r = 0.434; P < 0.05), parenchymal cell volume (r = 0.574; P < 0.001), and most strongly with total hepatocyte number (r = 0.614; P < 0.001) in all patients. Significant correlations were also observed between these three parameters and antipyrine clearance (r = 0.367, P < 0.05; r = 0.663, P < 0.001; and r = 0.807, P < 0.001, respectively). The mean aminopyrine clearance per individual hepatocyte showed no significant difference between cirrhotic and noncirrhotic patients (3.52 ± 1.60 × 10-10 mL/min vs. 3.65 ± 1.50 × 10-10 mL/min, respectively; P > 0.10). Similar results were obtained for antipyrine clearance per hepatocyte (7.35 ± 2.27 × 10-10 mL/min for cirrhotics vs. 6.16 ± 1.07 × 10-10 mL/min for noncirrhotics; P > 0.10). Thus, the intrinsic clearances of drugs per individual hepatocyte, as originally proposed in the intact cell hypothesis, were directly evaluated for the first time, lending strong support to the intact hepatocyte theory.

Microvascular oxygen transport: impact of a left-shifted dissociation curve.

The impact of an increased hemoglobin oxygen affinity (decreased P50) on oxygen transport was evaluated in capillaries of the retractor muscle under nonhypoxic (FIo2 = 0.30 and 0.21) and hypoxic (FIo2 = 0.10) conditions in hamsters with normal oxygen affinity [control; P50 = 26.1 +/- 1.0 (SD) mmHg, n = 12] and in hamsters with an increased oxygen affinity [treated; P50 = 16.2 +/- 1.6 (SD) mmHg, n = 7] induced by chronic short-term administration of sodium cyanate. Using in vivo video microscopy and computer-aided image analysis, we determined oxygen saturation (SO2) and associated hemodynamic parameters in both arteriolar (n = 30 control, 18 treated) and venular (n = 25 control, 17 treated) capillaries. In response to hypoxia, systemic arterial PO2 decreased to 29.6 +/- 6.0 (SD) mmHg in control animals and 24.7 +/- 3.8 (SD) mmHg in treated animals associated with abrupt decreases in systemic arterial blood pressure and increases in respiratory rate. The decrease in SO2 across the capillary network during nonhypoxic ventilation was 13.3% SO2 for control animals and 11.0% SO2 for treated animals. During hypoxic ventilation, the decrease in SO2 was 9.1% SO2 in control animals and 8.7% SO2 in treated animals. Hemodynamic parameters were not significantly different in the two groups during hypoxia. Estimated end-capillary PO2 was significantly lower in the treated animals. These data indicate that an increased oxygen affinity does not provide an obvious advantage for oxygen transport during hypoxia at the level of the capillary network in resting striated muscle; however, such an advantage might become apparent in the presence of an increased metabolic rate or a more severe hypoxic challenge.

Regulation of ciliary movement in sea urchin embryos: Dopamine and 5-HT change the swimming behaviour

1. Effects of dopamine and 5-HT on the swimming behaviour of larvae of the sea urchins, Pseudocentrotus depressus and Hemicentrotus pukherrimus were investigated by means of computer-aided image analysis. 2. Dopamine reduced the speed of forward swimming of the larvae after prism stage, and induced backward swimming in plutei of P. depressus, but not in H. pulcherrimus. 3. 5-HT tended to increase the speed of forward swimming of plutei and suppress spontaneous backward swimming.

In situ detection of autoanti-idiotype antibody-forming cells induced by influenza virus infection

In situ immunocytochemical-staining methods combined with computer-aided image analysis were employed to examine autoanti-idiotype antibody-forming cell expansion in vivo. Autoanti-idiotype antibody-forming cells were demonstrated in the spleens of C57BL/6J (B6) strain mice intranasally infected with the influenza virus A/Hong Kong/168/(H3N2)[R]X-31. Autoantiidiotype B cells were detected and elevated in spleen tissues after secondary influenza infections compared to normal B6 mice, and were specific for a dominant idiotype antibody called PY206 reactive with the influenza virus hemagglutinin. Influenza virus-infected BALB/c mice, which make little PY206 Id, did not have increased autoanti-Id against PY206 compared to normal mice. Results provide evidence for an idiotype network-mediated stimulation of autoanti-idiotype B cell production during influenza virus infection.