Image Cytometric Analysis of Algal Spores for Evaluation of Antifouling Activities of Biocidal Agents.

Chemical biocides have been widely used as marine antifouling agents, but their environmental toxicity impose regulatory restriction on their use. Although various surrogate antifouling biocides have been introduced, their comparative effectiveness has not been well investigated partly due to the difficulty of quantitative evaluation of their antifouling activity. Here we report an image cytometric method to quantitatively analyze the antifouling activities of seven commercial biocides using Ulva prolifera as a target organism, which is known to be a dominant marine species causing soft fouling. The number of spores settled on a substrate is determined through image analysis using the intrinsic fluorescence of chlorophylls in the spores. Pre-determined sets of size and shape of spores allow for the precise determination of the number of settled spores. The effects of biocide concentration and combination of different biocides on the spore settlement are examined. No significant morphological changes of Ulva spores are observed, but the amount of adhesive pad materials is appreciably decreased in the presence of biocides. It is revealed that the growth rate of Ulva is not directly correlated with the antifouling activities against the settlement of Ulva spores. This work suggests that image cytometric analysis is a very convenient, fast-processable method to directly analyze the antifouling effects of biocides and coating materials.

Effect of phototherapy on B and T lymphocytes in Egyptian infants suffering from neonatal jaundice

BACKGROUND: Neonatal jaundice is one of the most common problems that affect newborn infants, and phototherapy is usually used for treatment. OBJECTIVES: Evaluation of the effect of phototherapy on neonatal immune system through measuring the percentage of B and T lymphocytes and determining the frequency of development of infections and need for hospitalisation during the first six months of life. METHODS: A prospective cohort study was conducted on 50 full term new-borns; 25 with indirect hyperbilirubinaemia and treated with conventional phototherapy and 25 healthy matched neonates as untreated controls. The percentages of CD19+, CD4+ and CD8+ lymphocytes were measured by flow cytometry before phototherapy and 72h after exposure. Follow-up of the study group for the occurrence of infections for a period of six months after phototherapy. RESULTS: The study showed a significant difference in CD19+ lymphocytes percentage between patients before phototherapy and controls (P value<0.01), also a significant correlation between serum levels of total bilirubin in patients and CD19+ lymphocytes percentage (P value<0.05). There was no significant difference between the percentages of CD19+, CD4+ and CD8+ lymphocytes in patients before or after 72h of exposure to phototherapy (P value>0.05). Also, there was no correlation between the percentages of CD19+, CD4+ and CD8+ lymphocytes after 72h of exposure to phototherapy and the occurrence of infections (Gastrointestinal tract and Respiratory tract infection) after six months of follow-up (P value>0.05). More studies are needed with larger number of patients to determine the effect of phototherapy on immune system

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High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry.

Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose-response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC50 value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA.

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA's distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA's ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

NeuriteQuant: an open source toolkit for high content screens of neuronal morphogenesis.

BACKGROUND: To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems. RESULTS: Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology. CONCLUSIONS: NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.

High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device.

Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 µM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.

Photothermal and photoacoustic Raman cytometry in vitro and in vivo.

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump-Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420-2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level.

High-content, image-based screening for drug targets in yeast.

BACKGROUND: Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast. METHODOLOGY: In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants. CONCLUSIONS: Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.

A systematic random sampling scheme optimized to detect the proportion of rare synapses in the neuropil.

Synapses can only be morphologically identified by electron microscopy and this is often a very labor-intensive and time-consuming task. When quantitative estimates are required for pathways that contribute a small proportion of synapses to the neuropil, the problems of accurate sampling are particularly severe and the total time required may become prohibitive. Here we present a sampling method devised to count the percentage of rarely occurring synapses in the neuropil using a large sample (approximately 1000 sampling sites), with the strong constraint of doing it in reasonable time. The strategy, which uses the unbiased physical disector technique, resembles that used in particle physics to detect rare events. We validated our method in the primary visual cortex of the cat, where we used biotinylated dextran amine to label thalamic afferents and measured the density of their synapses using the physical disector method. Our results show that we could obtain accurate counts of the labeled synapses, even when they represented only 0.2% of all the synapses in the neuropil.

Long-term astroglial reaction and neuronal plasticity in the subcortical visual pathways after a complete ablation of telencephalon in pigeons (Columba livia).

This paper analyzes the astroglial and neuronal responses in subtelencephalic structures, following a bilateral ablation of the telencephalon in the Columba livia pigeons. Control birds received a sham operation. Four months later the birds were sacrificed and their brains processed for glial fibrillary acid protein (GFAP) and neurofilament immunohistochemistry, markers for astrocytes and neurons, respectively. Computer-assisted image analysis was employed for quantification of the immunoreactive labeling in the nucleus rotundus (N.Rt) and the optic tectum (OT) of the birds. An increased number of GFAP immunoreactive astrocytes were found in several subregions of the N.Rt (p< .001), as well as in layers 1, 2cd, 3, and 6 of the OT (p< .001) of the lesioned animals. Neurofilament immunoreactivity decreased massively in the entire N.Rt of the lesioned birds; however, remaining neurons with healthy aspect showing large cytoplasm and ramified branches were detected mainly in the periphery of the nucleus. In view of the recently described paracrine neurotrophic properties of the activated astrocytes, the data of the present study may suggest a long-lasting neuroglial interaction in regions of the lesioned bird brain far from injury. Such events may trigger neuronal plasticity in remaining brain structures that may lead spontaneous behavior recovery as the one promoted here even after a massive injury.

Electroacupuncture decreases nitric oxide synthesis in the hypothalamus of spontaneously hypertensive rats.

Acupuncture-related effects on autonomic function have been explored via biological and neurophysiologic studies. The hypothalamus, known to regulate the autonomic nervous system, is likely affected by acupuncture treatment that modulates sympathetic functions. The aim of this study was to investigate the effect of electroacupuncture at the Jogsamni point (ST36, an acupoint known to modulate autonomic function) on expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus of spontaneously hypertensive rat. Nitric oxide, which is produced by nNOS activity, plays an important role in the regulation of many physiologic processes, including sympathetic activities, in the hypothalamus and other parts of the brain. nNOS expression was assessed by immunohistochemistry of nNOS and histochemistry of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). The staining intensities of nNOS-positive neurons and NADPH-d-positive neurons were quantitatively assessed using microdensitometry to measure changes in optical density. The results show that electroacupuncture at ST36 reduced the expression and activity of nNOS in the hypothalamus of spontaneously hypertensitive rats. These findings suggest that the electroacupuncture at ST36 results in modulation of the activity of nNOS in the hypothalamus of spontaneously hypertensive rat.

Morphometric analysis of the AMPA-type neurons in the Deiter's vestibular complex of the chick brain.

Chicken (Gallus gallus) brains were used to investigate the typology and the immunolabel pattern for the subunits composing the AMPA-type glutamate receptors (GluR) of hindbrain neurons of the dorsal (dND) and ventral nuclei (vND) of the Deiter's vestibular complex (CD), which is the avian correspondent of the lateral vestibular nucleus (LVN) of mammals. Our results revealed that neurons of both divisions were poor in GluR1. The vND, the GluR2/3+ and GluR4+ label presented no area or neuronal size preference, although most neurons were around 75%. The dND neurons expressing GluR2/3 are primarily around 85%, medium to large-sized 85%, and predominantly 60% located in the medial portion of the rostral pole and in the lateral portion of the caudal pole. The majority of dND neurons containing GluR4 are also around 75%, larger (70% are large and giant), exhibiting a distribution that seems to be complementary to that of GluR2/3+ neurons. This distinct arrangement indicates functional differences into and between the DC nuclei, also signaling that such variation could be attributed to the diverse nature of the subunit composition of the GluRs. Discussion addresses the morphological and functional correlation of the avian DC with the LVN of mammals in addition to the high morphological correspondence, To include these data into the modern comparative approach we propose to adopt a similar nomenclature for the avian divisions dND and vND that could be referred as dLVN and vLVN.

Reduced infarct volume and differential effects on glial cell activation after hyperbaric oxygen treatment in rat permanent focal cerebral ischaemia.

Permanent middle cerebral artery occlusion (MCAO) causes neurodegeneration and a robust activation of glial cells primarily in sensorimotor brain regions of rats. It has been shown that hyperbaric oxygen (HBO) increases oxygen supply to ischaemic areas and reduces neuronal cell loss. The effects of HBO treatment on microgliosis and astrogliosis in permanent cerebral ischaemia have not been addressed so far, but might be critical for neurodegeneration and neuroprotection, respectively. Therefore, we used spontaneously hypertensive rats with permanent MCAO to investigate the time window to start HBO and to compare the effects of different HBO treatment frequencies on infarct volume and on differences with regard to microgliosis and astrogliosis. Seven days after MCAO the infarct volume was calculated from Nissl-stained brain sections by image analysis. HBO significantly decreased the infarct volume when used as early as 15, 90 or 180 min post-MCAO by 24%, 16% and 13%, respectively, in the single-treatment group. Repetitive HBO treatment (first HBO session 90 min after MCAO) was not effective. Microglial cells and astrocytes were detected by cytochemical fluorescent labelling and confocal laser scanning microscopy. In the single-treatment group we observed significantly higher astrocyte immunoreactivity but decreased microglial density in the peri-infarct region. These effects of HBO treatment on glial cells were not present in rats where HBO did not reduce the infarct volume (360 min after MCAO). Our data indicate that HBO-induced suppression of microgliosis and aggravated response of astrocytes might contribute to the reported beneficial effects of early HBO treatment in cerebral ischaemia.

An oscillating extracellular voltage gradient reduces the density and influences the orientation of astrocytes in injured mammalian spinal cord.

We have studied the cellular basis for recovery from acute spinal cord injury induced by applied electric fields. We have emphasized this recovery is due to the regeneration of spinal axons around and through the lesion, and have begun to evaluate the contribution of other cells to the recovery process. We have imposed a voltage gradient of about 320 microV/mm across puncture wounds to the adult rat spinal cord in order to study the accumulation and orientation of GFAP+ astrocytes within and adjacent to the lesion. This electric field was imposed by a miniaturized electronic implant designed to alternate the polarity of the field every 15 minutes. Astrocytes are known to undergo hyperplastic transformation within injured mammalian cords forming a major component of the scar that forms in response to injury. We have made three observations using a new computer based morphometry technique: First, we note a slight shift in the orientation of astrocytes parallel to the long axis of the spinal cord towards an imaginary reference perpendicular to this axis by approximately 10 degrees--but only in undamaged white matter near the lesion. Second, the relative number of astrocytes was markedly, and statistically significantly, reduced within electrically--treated spinal cords, particularly in the lesion. Third, the imposed voltage gradient statistically reduced the numbers of astrocytes possessing oriented cell processes within the injury site compared to adjacent undamaged regions of spinal cord.

Estudio del nevus displásico mediante citometría de imagen / Dysplastic nevi by image cytometry study

Hemos realizado un estudio de la ploidía de ADN de 25 nevus displásicos (ND) mediante análisis de imagen computarizado. Los ND fueron seleccionados previamente desde el punto de vista clínico, e histopatológicamente se valoró el grado de atipia celular y arquitectural siguiendo las recomendaciones de la European Organization for Research and Treatment of Cancer Cutaneous (EORTC).Se han seleccionado 200 núcleos neoplásicos de la zona de mayor espesor tumoral, determinándose el área nuclear, densidad óptica integrada (DOI), índice de ADN (IADN), y valor de ploidía mayor de 5c (5cER), y se ha realizado estadística analítica y descriptiva con los resultados obtenidos. Hemos encontrado cierta asociación entre aneuploidía y atipia citológica, si bien, no hemos hallado diferencias significativas en los parámetros estudiados en el ND (AU)

Neural network prediction of prostate tissue composition based on magnetic resonance imaging analysis. A pilot study.

OBJECTIVE: To develop a neural network model that estimates prostate histology using magnetic resonance imaging (MRI). STUDY DESIGN: Fifty-three men with lower urinary tract symptoms (average age = 63.8 +/- 8.9 years) underwent a prostate MRI (T2) and sextant biopsy of the prostate. Masson Trichome and immunohistochemical prostate-specific antigen staining of the biopsy material were used to calculate the amount of stroma and epithelium in the inner gland (central plus transition zone). MRIs were normalized to the mean intensity of the obturator internus muscle for comparative analyses. Gray scale and texture features were extracted from the inner gland in the midsection transverse MRI slice. Clinical and image variables were used in two neural networks predicting a high amount of stroma and a high amount of epithelium, respectively. RESULTS: The positive and negative predictive values of the stroma and epithelium neural networks were 95%, 69% and 65%, 92%, respectively. CONCLUSION: These data suggest that the combined use of these neural networks may predict patient response to medical therapy targeting prostatic stroma or epithelium.

Use of three-dimensional microscopy with multiple oblique illumination in evaluating cytopathology preparations.

OBJECTIVE: To analyze whether the use of multiple oblique illumination (MOI), in contrast to axial illumination (AI), improves imagery in bright field microscopy. Specimens containing thick cell clusters, such as cervical Pap smears, are often misread because of out-of-focus cell clusters. We hypothesized that visualization of these specimens with MOI would enhance this information as compared with AI. STUDY DESIGN: Micrometer images and Pap smears were analyzed in focus, and 8 and 40 microns out of focus by MOI and AI. All fields were captured to a remote computer, histograms were constructed, and mean light intensity was calculated. Mathematical formulation was used to define the histogram distribution of the micrometer images. Statistical analysis was performed using one-way ANOVA and Newman-Keuls test. RESULTS: K(focus) was improved (P < .01) and at 20 and 40 microns out of focus, mean light intensities were greater (P < .003, P < .05, respectively) with MOI as compared to AI. CONCLUSION: MOI improves out-of-focus information by increasing light penetration through the specimen and enhancing contrast and resolution.

Morphometric analysis of human gingival elastic fibres degradation by human leukocyte elastase protective effect of avocado and soybean unsaponifiables (ASU).

Degradation of preelastic fibres (oxytalan and elaunin) and mature elastic fibres by human leukocyte elastase (HLE) was investigated using automated image analysis. Specimens from two young healthy adults were used. Although HLE hydrolyzed both fibre types, mature elastic fibres exhibited greater susceptibility to this effect than preelastic fibres. Avocado and soybean unsaponifiables are widely prescribed in rheumatology and parodontology and have also been the focus of ex vivo experiments aimed at determining whether they protect elastic fibres against degradation by HLE. Findings from the present study indicate that avocado and soybean unsaponifiables protect all types of gingival elastic fibres from degradation by HLE. Avocado and soybean unsaponifiables may be beneficial in patients with gingival inflammation and parodontitis, since HLE plays a major role in these disease states.

Automated histometry in quantitative prostate pathology.

OBJECTIVE: To review progress on the development of machine vision and image understanding in prostate tissue histology and to discuss the problems and opportunities afforded to pathology through the use of these techniques. STUDY DESIGN: A variety of concepts in machine vision are explored, and methodologies are described that have been developed to deal with the complexities of histologic imagery. The theory of human vision and its impact on machine vision are discussed. Software has been specifically developed for the analysis of prostate histology, allowing accurate gland segmentation, basal cell identification and measurement of vascularization within lesions. RESULTS: Image interpretation can be achieved using knowledge-based image analysis and the application of local object-oriented processing. This successfully allows an automated quantitative analysis of histologic morphology in the diagnosis of prostate intraepithelial neoplasia and invasive prostatic cancer. The use of low-power image scanning, based on textural or n-gram mapping, permits the development of fully automated devices for the rapid detection of tissue abnormalities. High-power, knowledge-guided scene segmentation can be carried out for the quantitative analysis of cellular features and the objective grading of the lesion. CONCLUSION: Automated tissue section scanning and image interpretation is now possible and holds much promise in prostate pathology and other diagnostically demanding areas. Issues of standardization still need to be addressed, but the development of such systems will undoubtedly enhance our diagnostic capabilities through the automation of time-consuming procedures and the quantitative evaluation of disease processes.

Quantification of tissue eosinophils and lymphocytes in histologic sections.

During a study of eosinophil-predominant gallbladder disease, an image analysis (IA) technique was developed for quantification of eosinophils and lymphocytes in routine formalin-fixed tissue sections. Alternating sections were stained with hematoxylin and eosin for eosinophils and with monoclonal CD45 antibody visualized with diaminobenzidine by an avidin-biotin procedure for lymphocytes. A protocol was then developed using a commercially available image analyzer and two well-defined macro routines. The system was validated with cell block sections prepared from peripheral blood samples with known eosinophil and lymphocyte counts. The eosinophil counts obtained by this IA technique showed excellent correlation with the absolute counts from a peripheral blood analyzer (r2 = 0.987). The lymphocyte counts obtained by IA showed good correlation with the absolute counts (r2 = 0.820). This IA-based technique provides a sensitive, reproducible, and substantiated means of quantifying inflammatory cells in tissue sections. This rapid and easily learned technique adds quantification as a complementary dimension to the subjective assessment of tissue morphology.