ImageStream System Research Articles

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Face Recognition using Fast GA

Face Identification System using a fast genetic algorithm computation (FGA) is presented. FGA is used to compute and search the face in a database. The objective of the work is to make a face identification system which can recognize face from a given image or any other image streaming system like webcam. The system also has to detect the face from a system accurately in order to identify the face accurately. The image can be captured either from a proposed webcam or a captured JPEG or PNG image or any other data source. The system needs training with adequate sample images to perform this operation. Training the generic system plays a vital role in identifying the face in an image. A tolerance is identified as a limit to the genetic algorithm which acts as a terminal condition to the evolution. A unique encoding is used which stores the facial features of a human face into numeric string which can be stored and searched with much ease thereby decreasing the search and computational time. Template matching technique is applied to identify the face in a big picture. Generation of an Eigen face is obtained by the stage a mathematical practice called PCA. Eigen Features is also computed such that the measurement of facial metrics is done using nodal point measurement.

Gelatin Based Polymer Cell Coating Improves Bone Marrow-Derived Cell Retention in the Heart after Myocardial Infarction.

Acute myocardial infarction (AMI) and the ensuing ischemic heart disease are approaching an epidemic state. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Polymer based cell coating is biocompatible and has been shown to be safe. Here, we assessed the therapeutic utility of gelatin-based biodegradable cell coatings on bone marrow derived cell retention in ischemic heart. Gelatin based cell coatings were formed from the surface-mediated photopolymerization of 3% gelatin methacrylamide and 1% PEG diacrylate. Cell coating was confirmed using a multimodality approach including flow cytometry, imaging flow cytometry (ImageStream System) and immunohistochemistry. Biocompatibility of cell coating, metabolic activity of coated cells, and the effect of cell coating on the susceptibility of cells for engulfment were assessed using in vitro models. Following myocardial infarction and GFP+ BM-derived mesenchymal stem cell transplantation, flow cytometric and immunohistochemical assessment of retained cells was performed. Coated cells are viable and metabolically active with coating degrading within 72h in vitro. Importantly, cell coating does not predispose bone marrow cells to aggregation or increase their susceptibility to phagocytosis. In vitro and in vivo studies demonstrated no evidence of heightened immune response or increased phagocytosis of coated cells. Cell transplantation studies following myocardial infarction proved the improved retention of coated bone marrow cells compared to uncoated cells. Gelation based polymer cell coating is biologically safe and biodegradable. Therapies employing these strategies may represent an attractive target for improving outcomes of cardiac regenerative therapies in human studies.

Evaluation of the Effect of Variations in Vehicle Velocity and Channel Bandwidth on an Image-Streaming System in Vehicular Networks

In this paper, we propose a solution for implementing a real-time image-streaming system for vehicle networks. Our proposed system organizes each vehicle as a local Internet of Things network. In e...

Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF.

Abstract 4476: Targeted delivery of curcumin to improve therapeutic outcome in breast cancer

Abstract Conventional chemotherapeutics offered to treat breast cancer (BrCa) are not selectively targeted. Due to this fact, they are toxic to the normal cells. It is imperative to identify new anti-cancer agents and/or methods to selectively target BrCa cells without affecting normal cells. In this regard, Curcumin, a plant polyphenol, has shown very promising results. Despite the proven anti-tumor effects of curcumin, clinical use of this compound is restricted due to its poor bioavailability. To surmount these problems, we developed nanoparticles, loaded with curcumin, using a planetary-balled milling (PBM) technique with particles tagged with folate for selective targeting to BrCa cells, which express high affinity folate receptors. Multispectral imaging, using an Amnis ImageStream system, was used to measure the response of curcumin-loaded PBM nanoparticles, and free curcumin on various BrCa cell lines. Curcumin-loaded folic acid-coated PBM nanoparticles selectively targeted and were taken-up by BrCa cells and induced apoptosis. Furthermore, our results indicate that targeted delivery of curcumin using PBM nanoparticles was 60 % more effective in killing BrCa cells at concentrations lower than the IC50 of free curcumin. Citation Format: Rajesh Singh, James W. Lillard. Targeted delivery of curcumin to improve therapeutic outcome in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2014-4476

Abstract 19198: Adult Human Heart Contains Two Populations of C-kit-Oct-4+SSEA-4+ Very Small Embryonic-Like Stem Cells (Isl-1- and Isl-1+)

Very small embryonic-like cells (VSELs) express pluripotent stem markers (Oct-4,Nanog, SSEA-4). and in adult mice may be expanded and differentiatiated into three germ layers. In humans VSELs were found in cord blood and peripheral blood (PB). AIM: Comparison of VSELs isolated from human myocardium with cardiac stem cells (CSC) and to measure the mobilization into PB after cardiac surgery. METHODS: 32 patients undergoing CABG were enrolled. Right atrial appendage (RAA), left ventricle (LV) were collected. PB samples were drawn before and immediately after surgery, 24 hrs and 5-7 days after weaning from cardiopulmonary bypass. Tissue samples (0.5-1.0 cm3) were cut, digested in collagenase, passed through 70um cell strainer, centrifugated, resuspended and stained with antibodies CD34 FITC/CD133 APC/SSEA-4PE/CD45 APC-Cy7. Analyses were carried out on Image Stream System which is a combination of FACS and immunofluorescent microscope. RESULTS: VSELs and CSC were identified in LAA. VSELs had markedly smaller diameter (4.7 ± 0.2µm vs. 7.7 ± 0.4µm), were c-kit negative, expressed markers of pluripotent cells (Oct-4, SSEA-4) and CD133. Two populations of VSELs - both Isl-1 negative and positive were identified. Subpopulation of Oct-4+SSEA-4+Isl+ VSELs contained cells of intermediate size (5.7 ± 0.7µm) and might form more mature population of VSELs progenitors. VSELs undergo significant mobilization into PB following the CABG. CONCLUSION: Human myocardium in adults contains two subpopulations of Oct-4+SSEA4+ VSELs which are distinct from CSC. Cardiac surgery with cardiopulmonary bypass induced mobilization of VSELs into peripheral blood.

Expression and histopathological correlation of CCR9 and CCL25 in ovarian cancer

Ovarian carcinoma is the most lethal gynecological malignancy among women and its poor prognosis is mainly due to metastasis. Chemokine receptor CCR9 is primarily expressed by a small subset of immune cells. The interactions between CCL25 and CCR9 have been implicated in leukocyte trafficking to the small bowel, a frequent metastatic site for ovarian cancer cells. We have previously shown that ovarian cancer cells express CCR9 and play an important role in cell migration, invasion and survival in the presence of its natural ligand in vitro. In this study, we have evaluated the expression of CCR9 and CCL25 in ovarian cancer cells and clinical samples. Ovarian cancer tissue microarrays from University of Alabama at Birmingham and AccuMax were stained for CCR9 and CCL25. Aperio ScanScope was used to acquire 80X digital images and expression analysis of CCR9 and CCL25. Flow cytometry and the Image stream system were used to conform the expression of CCR9 and CCL25 in ovarian cancer cells. Our results show significantly higher (p<0.001) expression of CCR9 and CCL25 in serous adenocarcinoma followed by serous papillary cystadenoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, cystadenoma, mucinous boderline adenocarcinoma, clear cell carcinoma, granulosa cell tumor, dysgerminoma, transitional cell carcinoma, Brenner tumor, yolk sac tumor, adenocarcinoma and fibroma cases, compared to non-neoplastic ovarian tissue. Similar to tissue expression, CCR9 was also significantly expressed by the ovarian cancer cell lines (OVCAR-3 and SK-OV-3) in comparison to normal adult ovarian epithelial cell. We provide the first evidence that CCR9 and its natural ligand CCL25 are highly expressed by ovarian cancer tissue and their expression correlates with histological subtypes. Expression of this chemokine receptor and its ligand CCL25 within primary tumor tissue further suggests a potential role of this chemokine-receptor axis in ovarian cancer progression.

Mobilization of Bone Marrow-Derived Oct-4+ SSEA-4+ Very Small Embryonic-Like Stem Cells in Patients With Acute Myocardial Infarction

This study sought to assess of the mobilization of nonhematopoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction (MI). Acute MI induces mobilization of bone marrow stem cells. Recently, a rare population of VSELs, expressing markers of embryonic pluripotent stem cells (PSCs), was identified in adult murine bone marrow and human umbilical cord blood. Thirty-one patients with acute MI and 30 healthy subjects were enrolled. Blood was sampled on admission, after 24 h, and 5 days later. Erythrocytes were lysed and lin(-)CD45(-) VSELs were isolated using a live cell sorting system (FACSAria, Beckton Dickinson, San Jose, California). In healthy subjects the median number of circulating VSELs was very low (median 0.8 [range 0 to 1.3]) cells/microl. In acute MI, VSELs were mobilized early (median 2.7 [range 0.2 to 3.9] cells/microl; p < 0.001) and remained elevated after 24 h and 5 days (median 4.7 [range 0.2 to 6.4] cells/microl; p < 0.003, and median 2.6 [range 0.3 to 3.6] cells/microl; p < 0.03, respectively). The mobilization of VSEL was significantly reduced in patients older than 50 years and with diabetes in comparison with younger and nondiabetic patients. Circulating VSELs were small (7 to 8 microm) and enriched in the messenger ribonucleic acid of PSC markers (Oct-4, Nanog), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C), and endothelial (VE-cadherin) markers. The presence of PSC markers (Oct-4, SSEA-4) and the chemokine receptor CXCR4 in circulating VSELs was confirmed at the protein level by immunofluorescent staining and ImageStream system (Amnis Corporation, Seattle, Washington) analysis. Acute MI induced mobilization of VSELs expressing pluripotent markers, early cardiac and endothelial markers, and chemokine receptor CXCR4.

Automated Classification of Normal and Leukemic B Cells on the ImageStream® Imaging Flow Cytometer Using Immunocytomorphological Image Characteristics

Leukemic B cells can be distinguished from normal B cells based on their surface staining patterns and their morphology, but the differences can be subtle and the abnormal events rare. In order to reliably differentiate normal from leukemic B cells, objective quantitation of large data sets is required. This is accomplished with the ImageStream cytometer, which automatically acquires multispectral images of thousands of individual cells in flow at very high speeds. The associated IDEAS data analysis software measures hundreds of photometric and morphometric features per cell, thereby enabling simultaneous immunophenotyping and morphology-based measurements. The features quantify individual image characteristics such as shape, size, signal strength and texture. We have leveraged these features by implementing an automated classification routine that identifies the feature combination that best separates different cell types of interest. Our automated classification routine utilizes a modification of Fisher's Linear Discriminant criterion to measure discrimination power between two or more populations of cells. The classifier generates a composite feature consisting of a weighted linear combination of basic image features that provides maximum discrimination. Thus, our method transforms a multi-parameter classification task that can be difficult to conceptualize into a classification based on a single composite feature that can be readily conceptualized and visualized using a simple histogram output. Since the composite feature is generated from, and tested and optimized on, large numbers of cells, we obtain results that are highly objective, repeatable, statistically significant and scalable with data size. We used this classification approach to study normal and tumor cells of the hematopoietic lineage with the goal of improving diagnosis of hematological disorders. Here we show the results of using automated classification to distinguish the subtle differences between B cells of CLL patients and those of normal patients. We also used the classifier to distinguish B-cell ALL from B-cell CLL. In both cases, we used a combination of shape, size, texture and signal strength features from the SSC, brightfield and nuclear imagery generated by the ImageStream system. The data presented demonstrate the application of a novel automated cell classification method to the discrimination of normal and tumor cells within large and heterogeneous cell samples.

Clinical Evidence That Oct-4+ ssea-4+ Very Small Embryonic Like Stem Cells (VSEL) Are Mobilized into Peripheral Blood in Patients with Acute Myocardial Infarction (AMI): A Novel Prognostic Indicator

We demonstrated that murine bone marrow (BM) contains a mobile population of CXCR4+SSEA-1+Sca-1+lin CD45− very small embryonic like (VSEL) stem cells (Leukemia 2006: 20; 857) that are mobilized into peripheral blood (PB) in SDF-1 dependent manner in murine model of acute myocardiac infarct (AMI) (J Moll Cell Cardiol 2008, 44, 865–873). Since a similar population of CXCR4+CD133+CD34+SSEA-4+Oct-4+lin−CD45− cells resides also in human BM (Leukemia 2007: 21;297), we asked whether in humans, similarly as in mice, the AMI-related stress may trigger mobilization of these cells from BM into PB. To address this question, we evaluatedthe mobilization of VSEL in patients with AMI,measured expression of pluripotential stem cell (PSC), cardiac and endothelial markers in peripheral blood mononuclear cells and purified by FACS VSEL,and finally assessed the factors influencing mobilization of VSEL and correlation of cell number with left ventricular ejection fraction (LVEF).Totally, 31pts with AMI and 30 healthy subjects were enrolled. Peripheral blood was sampled on admission, after 24 hours and 5 days VSEL were enumerated by FACS and sorted by Facs-Aria. Presence of PSC markers (Oct-4, SSEA-4) and chemokine receptor CXCR4 in circulating VSEL was confirmed at the protein level immunofluorescent staining and ImageStream system (ISS) analysis. Plasma levels of SDF-1, HGF, VEGF, G-CSF were measured by ELISA. We noticed that in healthy subjects median number of circulating VSEL is very low [0.8 (0–1.3] cells/μL. In AMI VSEL were mobilized early [2.7 (0.2–3.9) cells/μL; p<0.001), and remained elevated after 24 hrs and 5 days [4.7 (0.2–7.4); p<0.001 and 2.6 (0.3–3.6) cells/μL; p<0.03, respectively]. Circulating VSELs were enriched in mRNA of PSC markers (Oct-4: 206±3-fold; Nanog: 282,1±4-fold), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C) and endothelial (VE-cadherin) markers. Highest relative expression level was detected after 24h after acute MI. VSEL size - cell diameter was approx. 7–8 μm. The mobilization of VSEL after 12 and 24 hrs was significantly higher in patients younger than 50y [4.9 (0–6.4) vs. 2.7 (0.2–5.1) cells/μL; P<0.05]. In patients with diabetes the number of mobilized VSEL after 12 and 24 hrs was significantly lower than in those without diabetes [1.9 (0–2.3) vs. 4.8 (0.2–6.2) cells/μL; P<0.05]. Mobilization of VSEL was impaired in patients with LVEF <40% in comparison to LVEF >40% [3.3 (0–4.9) vs. 4.7 (0.4–6.4) cells/μL; P<0.05]. Of note VSEL showed significant negative correlations with cardiac necrosis markers (maximum activity of CK-MB and troponin I) after 12 and 24 hrs, but not 5 days (R=−0.41, R=−0.45). Thus, for the first time in humans we provide evidence that AMI induces mobilization of VSEL expressing pluripotent markers as well SDF-1 binding receptor CXCR4. Mobilization of VSEL is impaired in older patients with diabetes and reduced LVEF. Number of mobilized VSEL is inversely correlated with myocardial necrosis markers and positively with SDF-1 levels.

“Decoding the Dots”: The ImageStream system (ISS) as a novel and powerful tool for flow cytometric analysis

Abstract The aim of this article is to provide a brief review of the ImageStream system (ISS). The ISS technology was developed as a novel method for multiparameter cell analysis and subsequently as a supportive tool for flow cytometry (FC). ISS integrates the features of FC and fluorescent microscopy collecting images of acquired cells for offline digital image analysis. The article presents an overview of the main characteristics of ISS and a comparison between ISS, FC and the laser scanning cytometer (LSC). We reviewed ISS applications focusing on those involved in cellular phenotyping and provide our own experience with using ISS as a supportive tool to classical FC and demonstrate the compatibility between FC and ISS photometric analysis as well as the advantages of using ISS to confirm FC results.

Morphological characterization of very small embryonic‐like stem cells (VSELs) by ImageStream system analysis

Recently, our group purified a rare population of primitive Sca1+/Lin−/CD45− cells from murine bone marrow by employing multiparameter cell sorting. Based on flow cytometric and gene expression analysis, these cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). In order to better characterize VSELs, we focused on their morphological parameters (e.g. diameter, nuclear to cytoplasmic ratio, cytoplasmic area) as well as expression of Oct-4. To examine the morphological features of VSELs, we employed a multi-dimensional approach, including (i) traditional flow cytometry, (ii) a novel approach, which is ImageStream (IS) cytometry and (iii) confocal microscopy. We demonstrate by all of the sensitive and precise methods employed, that VSELs are a population of very small cells, which are significantly smaller than haematopoetic stem cells (HSC) (3.63 ± 0.09 versus 6.54 ±0.17 μm in diameter). They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes. Besides confirming the size characteristics, confocal microscopic analysis also confirmed that VSELs express Oct-4, a marker of pluripotent embryonic stem cells. Morphological examination reveals that VSELs are unusually small eukaryotic cells that posses several characteristics of embryonic cells. Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes.

Extended depth of field imaging for high speed cell analysis

AbstractBackground:Fluoresence microscopy is an extremely useful tool to analyze the intensity, location and movement of fluorescently tagged molecules on, within or between cells. However, the technique suffers from slow image acquisition rates and limited depth of field. Confocal microscopy addresses the depth of field issue via “optical sectioning and reconstruction”, but only by further reducing the image acquisition rate to repeatedly scan the cell at multiple focal planes. In this paper we describe a technique to perform high speed, extended depth of field (EDF) imaging using a modified ImageStream® system whereby high resolution, multimode imagery from thousands of cells is collected in less than a minute with focus maintained over a 16 μm focal range.Methods:A prototype EDF ImageStream system incorporating a Wavefront Coded™ element was used to capture imagery from fluorescently labeled beads. Bead imagery was quantitatively analyzed using photometric and morphological features to assess consistency of feature values with respect to focus position. Jurkat cells probed for chromosome Y using a fluorescence in situ hybridization in suspension protocol (FISHIS™) were used to compare standard and Wavefront Coded‐based EDF imaging approaches for automated chromosome enumeration.Results:Qualitative visual inspection of bead imagery reveals that the prototype ImageStream system with EDF maintains focus quality over a 16 μm focus range. Quantitative analysis shows the extended depth field collection mode has approximately ten‐fold less variation in focus‐sensitive feature values when compared with standard imaging. Automated chromosome enumeration from imagery of Jurkat cells probed using the FISHIS protocol is significantly more accurate using EDF imaging.Conclusions:The use of EDF techniques may significantly enhance the quantitation of cell imagery, particularly in applications such as FISH, where small discrete signals must be detected over a wide focal range within the cell. © 2007 International Society for Analytical Cytology

Sensitivity measurement and compensation in spectral imaging

The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.