bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.
Authors: Schauenstein K , Schauenstein E , Wick G
Published: April 2011

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.
Flow microfluorometric analysis of herpesvirus infected BHK-21 and BALB/3T3 cell cultures.
Authors: Dunn J , Spizizen J , Meinke W
Published: April 2011

The interaction of herpes simplex type 1 with two eukaryotic cell lines (BHK-21 and BALB/3T3) was investigated by flow microfluorometric analysis after cell staining with mithramycin. Uninfected, lytically infected and persistently infected cell populations were examined. Viral replication within an infected cell population could be detected via FMF analysis. The low levels of viral replication occurring within persistently infected cell populations were also detectable. A marked degree of correlation was noted between morphological observations of infected cultures and the FMF profiles obtained. The results deomonstrate the great potential of this technique for the early detection and analysis of viral infection.
Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts.
Authors: Granström G , Linde A , Nygren H
Published: April 2011

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.
Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis.
Authors: Phillips Burnett C C , Anderson W A , Rüchel R
Published: April 2011

Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.
Retarded development of noenatal rat lung by maternal malnutrition.
Authors: Curle D C , Adamson I Y
Published: April 2011

Inadequate dietary intake during late pregnancy may have significant effects on the developing fetal lung which undergoes rapid cellular multiplication and differentiation shortly before birth. The morphology, glycogen distribution and acid phosphatase activity in normal and starved neonatal rats have been studied sequentially, by using histochemical and cytochemical methods. It has been shown that the normal pattern of lung growth and enzymatic development is retarded in neonates of malnourished mothers. A slowed rate of cellular division and differentiation in the critical prenatal period resulted in a more immature air-blood barrier at birth, with glycogen retention by some epithelial cells. Delayed Type 2 cell maturation with diminished acid phosphatase activity suggests a decrease in surfactant production in the malnourished newborn. In addition, fewer alveolar macrophages with reduced acid phosphatase activity were observed in the perinatal period of starved rats; this finding might have implications for the handling of inhaled bacteria shortly after birth. These results indicate that nutritional status of the mother has a marked effect on fetal lung growth and development by inhibiting cellular proliferation, differentiation and enzyme development by epithelial and macrophagic cells.
Relative mobility of con A and anionic receptor sites on the cell surface of lymphocytes. A quantitative-cytochemical study.
Authors: Bhalla D K , Perotti M E , Anderson W A
Published: April 2011

Events following the binding of Concanavalin A and cationized ferritin to the surface of normal human peripheral blood lymphocytes were followed using ultracytochemical methods. In this paper, we described a technique involving microdensitometry, which was employed in order to make a comparative cytochemical quantitation of the dense horseradish peroxidase-diaminobenzidine reaction products representing the states of Concanavalin A receptors under various conditions of distribution and redistribution. The technique presents a direct approach to the analysis of various probes that are currently being employed for studying the nature and distribution of various cell surface components. An attempt was also made to analyze the distribution of anionic sites, and their lateral redistribution is compared with the mobility of Concanavalin A receptors. The data presented suggests an independent mobility of receptors for Concanavalin A and cationized ferritin.
Immunocytochemical evidence for vasopressin receptors.
Authors: Castel M
Published: April 2011

An electron microscopic study was made of mouse pituitaries immunocytochemically stained with anti-lysine vasopressin (LVP) as the primary antiserum in the unlabeled antibody peroxidase-anti-peroxidase procedure. Vasopressin (VP) was identified in the neurosecretory granules of the neural lobe which stained with peroxidase anti-peroxidase molecules. Electron density was induced in secretory granules of the pars intermedia (PI), both in the melanocyte stimulated hormone and ACTH cell types, probably indicating VP molecules attached to binding (receptor) sites. Omission of anti-LVP abolished staining both in the neural lobe and the PL Anti-LVP absorbed with antigen, by admixing with LVP, abolished staining in the neural lobe but not in the PI; according to optical density measurements the PI showed a +/- 22% staining increase over controls. Staining intensity in the PI probably reflects occupancy of binding (receptor) sites for VP. Exposure of PI granules to LVP before the usual staining sequence resulted in +/- 48% increased staining. In water-deprived mice with high endogenous VP titers, staining was +/- 33% and +/- 40% more intense than in normal mice. Solid phase absorbed and eluted antibodies to LVP provided additional proof that staining in both neural lobe and PI could be attributed to anti-LVP. Results indicate that binding or receptor sites for VP are located on secretory granules in the PL Possible physiological significance is discussed.
Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas.
Authors: Mesa-Tejada R , Keydar I , Ramanarayanan M , Ohno T , Fenoglio C , Spiegelman S
Published: April 2011

An indirect immunoperoxidase method was first used to localize mouse mammary tumor virus (MMTV) antigens in paraffin sections of mammary tumors of Paris RIII and CD8F1 mice. By using the same method, an antigen with cross-reactivity to a group-specific antigen (gp52, a 52,000 dalton glycoprotein) of MMTV was detected in paraffin sections of human breast carcinomas. The specificity of this reaction with antibody against MMTV was examined by absorption of the IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and prufied gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 73 of 191 (38%) breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. A positive reaction of uncertain specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.
Adrenal luteinizing hormone releasing hormone receptors.
Authors: Bernardo L A , Petrali J P , Weiss L P , Sternberger L A
Published: April 2011

Paraffin sections of mouse adrenals processed with antiserum to luteinizing hormone-releasing hormone (LHRH) in the unlabeled antibody enzyme method reveal moderate staining in the cytoplasm of cells of zona fasciculata and reticularis. The stain is intensified upon pretreatment of sections with LHRH. Pretreated sections processed with solid phase immunoabsorbed LHRH are unstained. Analogues of LHRH deficient in the C-terminal glycine amide inhibit staining, while analogues deficient in the N-terminal pyroglutamic acid have no effect. It is concluded that the adrenal contains receptors for a ligand resembling LHRH in receptor and immunoreactivity. The possibility is considered that the ligand may be an inhibitor of pineal origin.
Steric hindrance as a factor in the reaction of labeled antibody with cell surface antigenic determinants.
Authors: Kent S P , Ryan K H , Siegel A L
Published: April 2011

The binding of rabbit anti-human IgG labeled with 125I, shellfish glycogen or ferritin to human IgG attached to the surface of rabbit RBC with chromic chloride was studied. Maximum binding was noted with 125I labeled antibody. Slightly but consistently less binding was found with shellfish glycogen labeled antibody. The binding of ferritin labeled antibody was strikingly reduced--usually one-third or less of that found with 125I labeled antibody alone. This suggests that under the conditions of these experiments, the attachment of large labels to antibody molecules results in reduced antibody binding to surface antigen. Steric hindrance is probably at least in part responsible for this reduced binding.
Immunoperoxidase localization of vitamin D dependent calcium binding protein.
Authors: Morrissey R L , Zolock D T , Bucci T J , Bikle D D
Published: April 2011

Calcium binding protein (CaBP) was localized by the indirect peroxidase-labeled antibody method in chick duodenum 72 hr after administering 32.5 nmol of cholecalciferol to vitamin D-deficient chicks. CaBP was observed in cytoplasm and nuclei of absorptive cells but was absent from goblet cells. Our results are consistent with the suggested functional role for CaBP in the prevention of intracellular accumulation of calcium by preventing mitochondrial accumulation of calcium, enhancing removal of calcium from absorptive cells, and/or preventing the "leaking" of calcium into cells through the lateral borders. They are not consistent with an extracellular functional role for CaBP.
An improved acriflavine-Feulgen reagent for quantitative DNA cytofluorometry.
Authors: Levinson J W , Langlois R G , Maher V M , McCormick J J
Published: April 2011

We have modified the acriflavine-Feulgen histochemical method for the quantitative determination of DNA by performing the staining of hydrolyzed cells affixed to cover slips with acriflavine dissolved in 90% ethanol. Compared to conventional techniques, in which the acriflavine is dissolved in aqueous HCl plus potassium metabisulfite, this method not only decreased non-Feulgen (background) staining in both the nucleus and the cytoplasm, but also increased the fluorescent intensity of Feulgen stained nuclei more than four-fold. Cells stained by our modified method exhibited a fluorescence maximum of 507 nm, which is similar to the 500 nm fluorescence maximum obtained with acriflavine bound to apurinic acid solution by a modification of the acriflavine-Feulgen method. These fluorescence maxima are in contrast to the 510 nm to 625 nm fluorescence maxima which are obtained when cells are stained according to conventional protocols. The fluorescence intensities of nuclei of synchronized cells stained by the modified method were proportional to DNA content. Thus, by the criteria of staining specificity, in situ and in solution fluorescence spectra agreement and quantitative staining, we conclude that our modified acriflavine-Feulgen method is more satisfactory for quantitatively measuring DNA in situ by cytofluorometry than the usual acriflavine-Feulgen method.
Palladium chloride as a stain for elastin at the ultrastructural level.
Authors: Morris S M , Stone P J , Rosenkrans W A , Calore J D , Albright J T , Franzeblau C
Published: April 2011

Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.
Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA.
Authors: Hayashi S , Gillam I C , Delaney A D , Tener G M
Published: April 2011

DNA in prepared chromosomes from the larval salivary glands of Drosophila melanogaster was hybridized with [125I]-labeled 5S and tRNA from the same organism. Autoradiography revealed that radioactivity was frequently bound to all regions of the slides, masking labeling of the chromosomes. Acetylation of the preparations before hybridization prevented the formation of this background and revealed the specific chromosomal sites.
Binding of deoxyribonuclease I to actin: a new way to visualize microfilament bundles in nonmuscle cells.
Authors: Wang E , Goldberg A R
Published: April 2011

Intracellular actin-containing fibers can be visualized by indirect immunofluorescence microscopy when they are stained with antibody directed against DNase I. The location of actin-containing fibers in cells appears to be similar to the staining pattern of antibody to actin. Actin fibers were also visualized by direct fluorescent microscopy with rhodamine-conjugated DNase I.
A thresholding method for automatic cell image segmentation.
Authors: Borst H , Abmayr W , Gais P
Published: April 2011

An algorithm for automatic segmentation of PAP-stained cell images and its digital implementation is described. First, the image is filtered in order to eliminate the granularily and small objects in the image which may upset the segmentation procedure. In a second step, information on gradient and compactness is extracted from the filtered image and stored in three histograms as functions of the extinction. From these histograms, two extinction thresholds are computed. These thresholds are suitable to separate the nucleus from the cytoplasm, and the cytoplasm from the background in the filtered image. Masks are determined in this way, and finally used to analyse the nucleus and the cytoplasm in the original image.
Computer aided analysis of chromatin network and basophil color for differentiation of mononuclear peripheral blood cells.
Authors: Harms H , Gunzer U , Aus H M , Rüter A , Haucke M , ter Meulen V
Published: April 2011

Computer aided differentiation of plasmoblasts, Pfeiffer cells, immunoblasts, lymphocytes and centrocytes is achieved with the parameters of chromatin network arrangement and structure, and multispectral cytoplasm color. The digital methods involve: (a) segmenting the nuclear image into topographic sections and analyzing the optical density distribution from the chromatin in these sections; (b) determining the nuclear structure with a 7 x 7 median filter, gradient filter and contour following algorithms; and (c) clustering two-dimensional chromatic data from panoptically stained cellular components. The parameters reported here are a subset of those needed for the automated diagnosis of many hematologic diseases especially the leukemias.
Effectiveness of feature groups for automated pairwise leukocyte class discrimination.
Authors: Kulkarni A V
Published: April 2011

Automated blood cell differentials using statistical classification techniques have been implemented in several commercial machines. The machine-derived features used to classify leukocytes resemble the descriptors used by humans performing visual classification, e.g. size, content, shape, color, and texture. However, because of our crude modeling of vision there is no universally accepted measure of characteristics such as shape, color, or texture. One expects, therefore, that features which are powerful discriminators for humans may perform poorly when quantified by machine, while other parameters may be more precisely measured automatically and so prove more useful in cell classification. This paper reports the results of a feature evaluation study for automated discrimination between all pairs of a large set of leukocyte classes consisting of both normal and abnormal types. To provide a framework for comparing automated feature ordering with the ranking attached by medical technologists, the machine-derived features were divided into six groups: size and content, mean and mode, cytoplasm/nucleus comparison, contrast and texture, color, and nuclear shape. A sequential procedure was used to select the best five-feature set from each group and the globally best five-feature set for each pairwise classification. The results illustrate the strengths and weaknesses of machine-derived descriptors for each type of decision.
Fluorescence intensity resolution in flow systems.
Authors: McCutcheon M J , Miller R G
Published: April 2011

The factor which can limit fluorescence intensity resolution in a flow cytometer of the type in which cells pass perpendicularly through a focussed laser beam depends on signal intensity. For the brightest sources (e.g. fluorescent DNA stains), the coefficient of variation (CV) is limited in our system to around 3% by stream hydrodynamics, unstable illumination intensity, nonstoichiometric staining, etc. The weakest detectable sources (e.g. fluorescent cell-surface labels) are limited in coefficient of variation by shot noise in the photomultiplier due to constant background light levels. Finally, over a fairly wide brightness range between these extremes, resolution is determined primarily by photoelectron statistical variation on the signal itself (i.e. "photon statistics"). Thus photon collection and detection efficiency (solid angle, barrier filter passband, detector quantum efficiency) become of primary importance.
A statistical approach to the classification of biological cells from their diffraction patterns.
Authors: Genter F C , Salzman G C
Published: April 2011

An effective method of graphically representing complex biological data is presented, and a statistical classification technique is outlined including an example involving exfoliated cervical cells.
A real-time delay monitor for flow-system cell sorters.
Authors: Martin J C , McLaughlin S R , Hiebert R D
Published: April 2011

For optimum performance in cell sorting, it is critical to assure proper timing in the charging of droplets to be deflected. A method for determining the transiet delay time in cell sorters has been devised and applied to daily operation in the Los Alamos sorter systems. This delay monitor relies on detection of either scattered or absorbed light from cells in the fluid stream near the point of droplet breakoff.
Nonparametric flow cytometry analysis.
Authors: Bagwell C B , Hudson J L , Irvin G L
Published: April 2011

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.
Signal analysis of slit-scan contour data.
Authors: Härtel W , Betz J W , von Sengbusch G , Bader P
Published: April 2011

As a method for the preselection of alarms in gynecological cell samples, the Battelle Cytophotometry Research Group uses the slit-scan technique to obtain various cell parameters, such as the N/C ratio and the relative DNA content, from fluorescently stained cells, which are aligned one-dimensionally in the tape system designed at Battelle. The system developed at Battelle Institute analyzes all signals that exceed the background noise. As the first step in processing the slit-scan data, several threshold levels permit the separation of various artifacts. In subsequent steps, the nuclear peak is recognized, the nuclear boundaries are calculated, and seven cell parameters are determined. For the alarm detection at present only one parameter, DNA fluorescence, is used for these determinations. Visual assignment of these data to definite objects on the tape makes it possible to obtain frequency distributions of: (a) all recorded objects within the sample on the tape; (b) all signals that are classified as cells; and (c) all types of objects that preferentially cause alarms.
Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells.
Authors: Leary J F , Todd P , Wood J C , Jett J H
Published: April 2011

In laser flow cytometry, an increasingly popular technique of analytical cytology, quantitative measurements of interest include cell and nuclear diameters. Electronic circuitry for a new cell sizing technique has been developed which measured the time that signal pulses from either fluorescence or light scatter sensors exceed a preset constant fraction of the peak signal amplitude (pulse width) or the time that it takes a signal to rise between constant fractions of the peak signal amplitude on the rising side of the pulse (pulse rise-time). These pulse width or pulse rise-time measurements were related to cell or nuclear diameters and were used in combination to determine nuclear size to cell size ratios. This method of sizing was found to be independent of fluorescent or light-absorbing stain intensity, linearly related to cell or nuclear diameter, and capable of resolving small diameter differences.
Simultaneous flow cytometric DNA and volume measurements of bone marrow cells as sensitive indicator of abnormal proliferation patterns in rat leukemias.
Authors: Valet G , Fischer B , Sundergeld A , Hanser G , Kachel V , Ruhenstroth-Bauer G
Published: April 2011

Simultaneous flow cytometric DNA and volume analysis of normal rat bone marrow cells shows three populations of nucleated cells with different mean volume. Each of these populations proliferates in a distinct cell cycle (alpha, beta, gamma). Normally the alpha-cell cycle has the highest amplitude, the beta-cell cycle is intermediate, and the gamma-cell cycle is low. The alpha-cell cycle was very significantly depressed and the beta + gamma-cell cycle was increased in three different rat leukemias (L5222, Shay, BNML), growing on three different rat strains (BDIX, Holtzmann, Brown Norway). The two parameter analysis further revealed that cells of the beta + gamma-cell cycle were slightly hyperdiploid and hypertetraploid in leukemic animals. The decrease of the alpha-cell cycle and the hyperploidies were more sensitive indicators for the abnormal proliferation pattern than the analysis of one parameter DNA distributions which remained within normal limits in all three leukemias.
Slit-scan flow cytometry of mammalian chromosomes.
Authors: Gray J W , Peters D , Merrill J T , Martin R , Van Dilla M A
Published: April 2011

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.
A screening system for cervical cancer cytology.
Authors: Golden J F , West S S , Shingleton H M , Můrad T M , Echols C K
Published: April 2011

A system for screening cervical cytological preparations is described which employs the Leitz Texture Analyzer System (E. Leitz, Rockleigh, N. J.) quantitative staining with acridine orange, and a fluorescence standard. The instrumentation scans cells on microscope slides and detects objects which it interprets to be nuclei with excess total nuclear green fluorescence intensity (Previous results employing manual measurements have indicated that normal nuclei do not produce total nuclear green fluorescence greater than a specific absolute intensity level). Detected objects are identified by visual observation. Cells (102,000) from 65 patients (29 normal, 36 abnormal) have been examined. In each abnormal sample, at least one abnormal cell was detected. In over half of the samples, three or fewer other objects (e.g. clumps of polymorphonuclear leukocytes) were detected. These are easily distinguishable from single nuclei, and could be discarded by someone with minimal cytological training.
Quantitative cytology of the positive region in flow sorted vaginal smears.
Authors: Goerttler K , Stöhr M
Published: April 2011

Fifty-one gynecologic specimens were collected from three women's hospitals and mailed in a prefixed status to our laboratory. The specimens were classified into a negative, a suspicious, a postradiation, and a positive group. After single cell dispersion the samples were stained for DNA and protein, analyzed, and sorted in the dual laser equipped Heidelberg flow analyzer sorter (HEIFAS). Particles with elevated DNA values (beyond 3.5 ploidy) and with intermediate protein values were sorted as the positive fraction directly on microscopic slides. After restaining according to Papanicolaou, they were re-evaluated cytologically and identified as tumor cells, dysplastic cells and false alarms. The latter consist of doublets and aggregates of more than two cells, binucleated cells, sperm aggregates and epithelial cells contaminated with bacteria. The different groups showed significant differences regarding the total rate of aggregates to single cells. In general, false alarms were very frequent in the positive region and impeded the statistical classification of the sample. The reduction of false alarms is a prerequisite for prescreening with flow instrumentation.
Interactive display and analysis of data from bivariate flow cytometers.
Authors: Bennett D E , Mayall B H
Published: April 2011

An interactive computer program, SWELL, displayss and analyzes bivariate distributions generated by flow cytometers. SWELL is modular with options available via a menu, is written in Fortran, and utilizes a video color display system. Data are accumulated as a bivariate distribution that is transferred to the computer as a 64 x 64 matrix. For ease of visualization, matrices are displayed in pseudocolor. The distribution values are broken into eight ranges and each range is represented by a color. Each element of the matrix is then displayed in its assigned color. To allow pooling and comparison, distributions are aligned, edited, and standardized. Unknown samples are pooled or analyzed singly and compared to the normal pool by subtraction. Differences are displayed as pseudocolor matrices of sign, magnitude, or statistical magnitude in units of standard deviation. This latter display, scaled to tolerance limits, readily reveals regions of significant difference between normal and abnormal samples. Counts within such regions can be compared to diagnose samples automatically.
Progress report of the TUDAB project for automated cancer cell detection.
Authors: Abmayr W , Burger G , Soost H J
Published: April 2011

Two methods for high resolution cell image data acquisition are applied routinely. Cells are either scanned by a computer controlled fast scanning microscope photometer (SMP) or a TV-camera. The software system for digital image analysis was completely revised and implemented on the PR 330 minicomputer. The system contains codes for primary cell data acquisition, segmentation of cells, cell feature extraction and statistical cell analysis. With this system, SMP and TV scanned cell data bases of PAP stained cells in vaginal smears, grouped into several classes, have been built up. Each data base contains 34 primary features and 20 feature combinations for each cell. A linear discriminant analysis is applied routinely for cell classification. The present state of the system and its operation are described, cell features and classification results are shown, and future steps for a prescreening strategy are discussed.
Light scattering and fluorescence by small particles having internal structure.
Authors: Kerker M , Chew H , McNulty P J , Kratohvil J P , Cooke D D , Sculley M , Lee M P
Published: April 2011

We consider two related, yet distinct queries: 1. How does the internal morphology of a small particle affect the elastic light scattering signals? We have devised an algorithm, presently accurate for particles comparable only to small biological spheres (diameter less than 1 micron), which suggests that light scattering is sensitive to internal morphology only in the backward directions. Accordingly, observations should be obtained in these directions when probing for internal morphology. 2. How are fluorescent signals affected when the active molecules are variously distributed within small particles? One cannot assume that the fluorescent signals are simply proportional to the number of active molecules contained in the particle because there may also be a dependence upon the geometrical and optical properties of the particle and upon the particular spatial distribution of these molecules within the particle. Indeed, even the measured emission spectrum may be affected by such morphological features. Here, too, these calculations are mainly restricted to small particles (diameter less than 1 micron) in which the fluorescent molecules are isotropic and immobile. Under these conditions the effects are quite dramatic. These effects should be considered in quantitative procedures which utilize fluorescence for determining the concentration of specific molecules in small particles such as biological cells. They may provide a clue for discriminating among cells which differ morphologically or in which the spatial distribution of the fluorescent moiety differs. These effects may be minimized by utilizing a light source which is polarized perpendicularly to the scattering plane.
Mass action and acridine orange staining: static and flow cytofluorometry.
Authors: Nicolini C , Belmont A , Parodi S , Lessin S , Abraham S
Published: April 2011

We present results involving an approach to acridine orange staining of intact cells based on basic physicochemical considerations. We show by static microfluorometry of several in vitro and in vivo cell lines that the important parameters for such staining are the molar ratio (Formula: see text), and molar concentration of acridine orange. Differential nuclear DNA and cytoplasmic RNA staining are totally controlled by these two parameters. We show this by a physicochemical model of cell-dye interaction. Finally, we use the method to study the growth parameters of complex in vivo cell populations by automated multiparameter flow microfluorometry. We have explored also, both by static and flow systems, the effect on AO-cell staining of various cell pretreatments such as Triton X-100 and chelating agents.
Sedimentation velocity separation: a preparation method for cervical samples.
Authors: Otto K , Höffken H , Soost H J
Published: April 2011

A preparation procedure, aiming at monolayer deposition of cervical exfoliative material on glass slides for high resolution prescreening has been developed. The main features of this procedure are centrifugal deposition after suspension and sedimentation of samples over isopycnic medium of 1.026 density. Fractioning of the separation column after centrifugation at 50 X g yields two preparations with leukocytes, bacteria and cellular debris predominantly located on the first slide and epithelial cells on the second one. The degree of spatial cellular isolation as well as the amount of diagnostically relevant cells per slide seem to fit the requirements of automated high resolution analysis.
A system for storage and retrieval of individual cells following flow cytometry.
Authors: Schildkraut E R , Hercher M , Shapiro H M , Young R E , Matsu N , Brown D C , Webb R H
Published: April 2011

A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.
Flow cytometry as a new method for the measurement of electrophoretic mobility of erythrocytes using membrane charge staining by fluoresceinated polycations.
Authors: Valet G , Bamberger S , Hofmann H , Schindler R , Ruhenstroth-Bauer G
Published: April 2011

The binding of FITC-labeled poly-L-ornithine and poly-L-lysine to fresh or neuraminidase treated human, rat or rabbit erythrocytes was investigated by simultaneous cell volume and cell membrane fluorescence measurements in a flow cytometer. The cell volume was converted into cell surface and the distribution curve of the fluorescence/micrometer2 cell surface was calculated from all histogram classes by a computer program. The mean fluorescence/micrometer2 cell surface as a measure of the density of the negative charges on the cell surface was directly proportional to the elctrophoretic mobility of the erythrocytes, showing that polycation binding can effectively be used for the measurement of the electrophoretic mobility of erythrocytes. The computer fitting of the experimental two parameter histograms by two dimensional Gaussian normal distributions was found to be a very efficient way of data reduction, and a good separation of overlapping cell clusters was possible even in the case of low total numbers of cells in the histogram.
Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.
Authors: Pinkel D , Dean P , Lake S , Peters D , Mendelsohn M , Gray J , Van Dilla M , Gledhill B
Published: April 2011

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.
Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.
Authors: Collste L , Darzynkiewicz Z , Traganos F , Sharpless T K , Whitmore W F , Melamed M R
Published: April 2011

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.
Quantitation of lymphocyte response to PHA by flow cytofluorometry. III. Heterogeneity of induction period.
Authors: Braunstein J D , Schwartz G , Good R A , Sharpless T K , Melamed M R
Published: April 2011

Early events in phytohaemagglutinin (PHA) stimulation of mouse splenocytes have been quantitated by using flow cytometry and supravital staining with acridine orange (AO). Increasing percentages of single cells with increased metachromatic (red) AO staining were demonstrated in cultures stimulated by PHA for up to 24 hr. These differences in staining could be eliminated by fixation with 1:1 ethanol/acetone before staining. Stimulated cells showed an increase in nonspecific esterase activity as measured by flow cytometry after supravital staining with fluorescein diacetate (FDA). The data reported show a heterogeneity in the per cell response of mouse splenocytes to PHA. The relationship between these data and the mechanism of mitogen stimulation is discussed.
BioPEPR: a system for the automatic prescreening of cervical smears.
Authors: Zahniser D J , Oud P S , Raaijmakers M C , Vooys G P , Van de Walle R T
Published: April 2011

A feasibility study has indicated that a Prescion Encoding and Pattern Recognition (PEPR) cathode ray tube prescreening system for cervical smears can be both accurate and fast. Smears are prepared using a syringing technique and are stained with a Feulgen-type nuclear stain and a protein counter-stain. The use of film as an intermediate step between the cells and Bio PEPR allows the scanning of fields as large as 8 x 8 mm. The morphological features of the cells are measured as directed by a hierarchical decision strategy. Additional programs detect artifacts, overlaps, and leukocytes. For clean samples, false positive and false negative rates on the cell level have been obtained that will allow acceptable smear level rates (10% false positive, 1% false negative). These rates have been reached without compromising the required speed goals of 120 to 180 smears per hr. The efficiency of the system is dependent on the quality of the smears. Measurements on a set of 192 routinely prepared smears indicate acceptable false negative rates and a false positive rate of about 18%. A reduction of this rate is expected with small improvements in cell preparation and measuring software, leading to the overall system efficiency required for commercial feasibility.
Interactions between pairs of DNA-specific fluorescent stains bound to mammalian cells.
Authors: Langlois R G , Jensen R H
Published: April 2011

The interactions between DNA-specific fluorescence stains complexed with mitotic Chinese hamster cells were studied by spectrofluorometric and flow fluorometric techniques. The degree of binding interactions and of energy transfer between stains was determined from the intensities and shapes of fluorescence emission spectra of cells complexed with pairs of stains. The stain pairs Hoechst 33258-chromomycin A3, Hoechst 33258-ethidium bromide, and chromomycin A3-ethidium bromide exhibited efficient energy transfer from the short wavelength absorber (donor) to the long wavelength absorber (acceptor), and little competitive or cooperative binding of stains. The stain pair quinacrine-ethidium bromide exhibited both energy transfer and competitive binding. None of the stain pairs showed evidence of strong electronic interactions between stains. The magnitude of energy transfer interactions was used to estimate the quantity and distribution of the stains molecules complexed to mitotic cells. The results indicate a fairly even distribution of each of these stains along the DNA of intracellular mitotic chromosomes.
An immunoferritin labeling study of H-2 antigens on dissociated epithelial cells.
Authors: Parr E L , Kirby W N
Published: April 2011

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from stomach, duodenum-jejunum, ileum, trachea, diestrus uterus, gall bladder, and vas deferens. Before cell dissociation, most of the organs were prefixed in periodate-lysine-paraformaldehyde to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. Five kinds of epithelial cells expressed H-2 antigens on lateral and basal membranes but not on apical membranes. These were the lining cells of the upper intestine, ileum, gall gladder, uterus, and the tracheal brush cell. The antigens were continuously distributed on the lateral and basal membranes of these cells and appeared to be absent from the apical membranes, rather than masked by the fuzzy coat. On four other epithelial cell types H-2 antigens could not be detected. These were the lining cells of the vas deferens, parietal and chief cells from the stomach, and ciliated tracheal cells. It does not seem to be uncommon for normal nucleated cells to lack H-2 antigens. On fixed and labeled epithelial cells from the upper intestine the zonula occludens membranes were unlabeled, while the zonula adherens and desmosome membranes were labeled as densely as the remainder of the lateral membranes. The zonula occludens membrane thus constituted the boundary betewen the unlabeled apical membrane and the labeled lateral membrane of these cells. Intestinal epithelial cells dissociated without prefixation showed a patchy distribution of H-2 antigens on their lateral membranes after indirect labeling, indicating antigen mobility in this membrane. On the same unfixed dissociated cells the antigens were able to migrate from lateral to apical membranes, a movement which appears to be prevented in the intact epithelial layer by the occluding junction. The absence of H-2 antigens from apical membranes and their inability to migrate through an intact zonula occludens suggest that these molecules must reach the lateral membranes of epithelial cells by a pathway which is distinct from that followed by apical membrane components.
Application of microspectrophotometry to biomedical research.
Authors: Grove G L
Published: April 2011

During recent years, the utilization of microspectrophotometry in biomedical research has increased at a rapid pace. This growth has been facilitated by both the advent of commercially available instruments which are reliable and manageable as well as improvements in histochemical techniques. This paper discusses the fundamental principles of visible light microspectrophotometry and important design criteria as well as briefly reviewing a wide range of applications. Special emphasis will be given to the Vickers M85 scanning integrating microspectrophotometer which is being routinely employed for both clinical and laboratory studies by a sizeable number of investigators.