Topology-based fluorescence image analysis for automated cell identification and segmentation

Cell segmentation refers to the body of techniques used to identify cells in images and extract biologically relevant information from them; however, manual segmentation is laborious and subjective. We present Topological Boundary Line Estimation using Recurrence Of Neighbouring Emissions (TOBLERONE), a topological image analysis tool which identifies persistent homological image features as opposed to the geometric analysis commonly employed. We demonstrate that topological data analysis can provide accurate segmentation of arbitrarily-shaped cells, offering a means for automatic and objective data extraction. One cellular feature of particular interest in biology is the plasma membrane, which has been shown to present varying degrees of lipid packing, or membrane order, depending on the function and morphology of the cell type. With the use of environmentally-sensitive dyes, images derived from confocal microscopy can be used to quantify the degree of membrane order. We demonstrate that TOBLERONE is capable of automating this task.     

Establishment of a persistent measles virus infection in HEp-2 cells.

A productive measles virus persistent infection has been established in HEp-2 cells. Greater than 90% of the persistently infected HEp-2 cells (H2MV) exhibited measles specific immunofluorescence and haemadsorption. Although most of the H2MV cells contained measles specific antigens, only a small percentage (less than 1%) actually produced infectious measles virus as determined by infectious centre assays. The measles virus produced by H2MV cells exhibited properties different from the initiating parent Edmonston strain virus, being reduced in virulence and also temperature sensitive for replication at 39 degrees C. The role of these altered virus properties in the establishment of persistence is considered.     

Context based mixture model for cell phase identification in automated fluorescence microscopy

Background  

Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task.     

Contextual analysis and intermediate cell markers enhance high-resolution cell image analysis for automated cervical smear diagnosis.

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.     

Intraspecies antagonism of Sh. flexneri in an HEp-2 cell line model]

The authors describe an effect of suppression of invasion of the guinea pig eye conjunctiva and the HEp-2 epithelial cells by virulent Sh. flexneri bacilli, with a simultaneous administration of the same dose of avirulent shigella mutants, genetically connected with them. The data of morphological study and experiments with 3H-glucose labeled shigellae carried out on the cell species model indicated that the bacterial competition for the specific sites for absorption on the epithelial cells underlay the observed phenomenon.     

Distribution of constitutive heterochromatin in Hela and HEp-2 cell lines

Summary Despite the fact that HeLa and HEp-2 cell lines have been established respectively from a female and a male patient, the frequent loss of the Y chromosome makes it difficult to discriminate the two cell types. The technique of centromeric localization of heterochromatin, however, facilitates more definite identification of the two cell types. Giemsa positive centromeric constitutive heterochromatin blocks were induced within the intact metaphase chromosome preparations of Hela and HEp-2 cells. The Hela cell population had a single heterochromatin marker chromosome, whereas the HEp-2 cell line was found to possess two types of heterochromatin markers. The distinctive patterns of distribution of constitutive heterochromatin of these three chromosomes served as the guides in tracing the presumptive cytological pathways for their orgin.

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Zusammenfassung Trotz der Tatsache, daß Hela- und HEp-2-Zellinien jeweils von einem weiblichen und einem männlichen Patienten stammen, erschwert der häufige Verlust des Y-Chromosoms eine Unterscheidung der beiden Zelltypen. Der Nachweis des zentromernahen Heterochromatins jedoch ermöglicht eine zuverlässigere Erkennung dieser beiden Zelltypen. Die Hela-Zellpopulation hat ein einzelnes, durch das Heterochromation gekennzeichnetes Marker-Chromosom, während die HEp-2-Zellinie deren 2 besitzt. Das distinkte Verteilungsmuster des konstitutiven Heterochromatons dieser 3 Chromosomen dient dazu, die vermutliche cytologische Herkunft zu interpretieren.

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Supported in part by the Robert A. Welch Foundation, The National Foundation —March of Dimes and USPHS Grant No. FR-05425.     

Specific-primer-directed DNA sequencing using automated fluorescence detection.

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.     

Quantitative analysis of dyes for automated studies of cell preparations

Spectral studies were made of the dyes in solutions and also of the cytology preparations stained by these dyes. 14 dyes of the blue-violet spectral region were examined. Absorbtion and transmission coefficients of the cell nucleus and cytoplasm were measured. The data obtained allowed to elaborate a quantitative estimation of dye selection. According to this method cresyl-violet was selected as the dye for Videocon LI-421.     

A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.     

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay.

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.     

Adenosine phosphorylase activity in a mutant HEp-2 cell line contaminated with Mycoplasm hyorhinis.

Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor conformycin. Isopycnic separation of [3H] thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [14C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified as Mycoplasma hyorhinis, a porcine mycoplasma. Following gamma-irradiation (3000 rads) to block cellular mitosis, the mucoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma.     

Components and results of a new preparation technique for automated analysis of cervical samples

Components and evaluations of a new preparative procedure for automated high-resolution analysis of cervical samples are presented. This procedure is based on sedimentation velocity separation of samples with subsequent fractionation of the separation column and centrifugal deposition of suspended cells on coated glass slides. A system for specimen collection and mailing of suspended samples is described. A new type of glass slide designed for automated analysis is presented, and centrifugal buckets for cell deposition on a 6-sq-cm area are described. Experimental results with different kinds of coating substances for glass slides as well as different isopyknic media are discussed, and data for differential cell counts are graphically demonstrated. Looking at the diagnostic accuracy and economic feasibility of this system, the authors realize that preparations have to be evaluated quantitatively and that constraints of sample size and processing time have to be taken into consideration for further developments.     

An automated method for DNA preparation from thousands of YAC clones.

We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library. Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate. The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR). Using this protocol, we produced 3000 YAC strain DNAs in three weeks. This automated procedure should be extremely useful in many genomic mapping projects.     

Multiparametric cell cycle analysis by automated microscopy

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.     

Dye free automated cell counting and analysis

We have developed an automated cell counting method that uses images obtained at multiple focal heights to enumerate cells in confluent culture. By taking the derivative of image intensity with respect to focal height using two complementary images, we are able to count high‐density monolayers of cells over a large image area. Our method resists errors arising from variability in the focal plane caused by flatness or tilt non‐uniformities with a minimal amount of focal plane alignment, allowing the automated collection of images across a large area. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.