共查询到20条相似文献,搜索用时 31 毫秒
1.
An epiillumination microscope objective slit-scan flow system has been fabricated utilizing two dimensional slit scanning with hydrodynamic sample stream focussing. Low resolution (4 micron) analysis of cellular fluorescence is facilitated by the definition of a stabilized flow plane through hydrodynamic focussing. Coincidence of the region of stabilized flow with the focal plane of the microscope objective will allow for the collection and subsequent imaging of fluorescence from cells oriented along this plane. Two orthogonal slit-scan contours are generated as a cell traverses the excitation region. It is hoped that the need for a three dimensional system will be precluded by preferential orientation of the cells in the region of stabilized flow. Cellular fluorescence is collected by a high numerical aperture epiillumination optical system and imaged onto two orthogonal slits. Two photomultiplier tubes are used to detect fluorescence. It is anticipated that the epiillumination microscope objective slit-scan flow system will be used with a variety of fluorescent stains and markers, as well as extended to the research of light scattered by cells. (Steen, H.B., Cytometry 1:26-31, 1980. 相似文献
2.
The usefulness of multidimensional slit-scan flow cytometry in whole cell measurements is dependent on extracting relevant features from the cellular fluorescence distributions (slit-scan contours). In addition, the extraction of these features must be rapid to allow for real-time data processing during acquisition. This paper describes two algorithms that have been used successfully to count the numbers of local maxima (peaks) and to find nuclear boundaries in a cellular fluorescence distribution. These routines are efficient, use only simple integer arithmetic, and have been implemented on several different microprocessors. 相似文献
3.
J E Gill L L Wheeless C Hanna-Madden R J Marisa 《The journal of histochemistry and cytochemistry》1979,27(1):591-595
Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes. 相似文献
4.
W H?rtel J W Betz G von Sengbusch P Bader 《The journal of histochemistry and cytochemistry》1979,27(1):311-314
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. 相似文献
5.
G von Sengbusch M Achatz R Beck J W Betz W H?rtel R Mann P Bader G Schlüter G Seger 《The journal of histochemistry and cytochemistry》1979,27(1):600-603
In this report, a preselection of alarms in a system for automated screening of cervical cancer based on depositing the cell sample linearly as a "cell trace" on a tape and analyzing it at different decision levels with increasing complexity, and preliminary results on analyzing cervical material with this system are discussed. The "cell trace" is analyzed with the slit-scan technique. Six parameters are computed: 1) cellular diameter; 2) nuclear diameter; 3) nuclear fluorescence (acriflavin-Feulgen) as nuclear DNA; 4) cellular fluorescence; 5) nuclear to cytoplasm ratio (N/C ratio); and 6) nuclear density. At present, only nuclear fluorescence is used to define a decision boundary between normal and potentially atypical cells. Under this criteria the slit-scan analysis leaves 5% of the events in a sample that must be rechecked at a second decision level in normal cell samples. A further reduction is expected when several slit-scan parameters are used at the first decision step. All events declared suspicious will be investigated in more detail by a two dimensional image analyzing system where the fluorescence image is generated by a laser scanning system. Results obtained in preliminary experiments are discussed in this paper. 相似文献
6.
M A Cambier W J Christy L L Wheeless I N Frank 《The journal of histochemistry and cytochemistry》1976,24(1):305-307
A study was undertaken to assess the applicability of the slit-scan technique to automated prescreening of urinary tract cytology. Cells from voided and catheterized urines were stained with acridine orange and measured on a static cell slit-scan cytofluorometer. Analysis of data from the specimens indicates that nuclear fluorescence alone appears adequate for recognition of abnormal specimens. Remaining problems in the automation of urinary tract cytology prescreening are discussed. 相似文献
7.
L L Wheeless D B Kay M A Cambier J L Cambier S F Patten 《The journal of histochemistry and cytochemistry》1977,25(7):864-869
False alarms, arising from a variety of sources, are the greatest remaining obstacle to development of an automated prescreening system for gynecologic cytology. This paper describes two correlation systems under development at the University of Rochester and discusses their utilization in the study of false alarms in slit-scan cytofluorometry. Both systems permit imaging of objects in flow and correlation between images and corresponding slit-scan contours. Correlation systems will permit a detailed study of false alarm causes and aid in the search for new features to assist in their recognition. 相似文献
8.
An erasable programmable read-only memory (EPROM) contour generator has been fabricated to produce contours for use in flow cytometry. Contours are analog waveforms representing the fluorescence or light-scatter intensity distribution along a cell or object. The generator has particular utility in the development and testing of slit-scan instrumentation and analysis algorithms. Contours are generated without the requirement of specimens or full operation of the flow instrumentation. The generator provides control of contour height, width, offset, and rate. The EPROM may be custom programmed to produce contours for specific test applications or for reproducing "real" contour events. The generator is useful in situations where constant repetitive contours of predetermined characteristics are required. 相似文献
9.
R C Mann 《Cytometry》1983,3(4):257-261
A model is presented to compare the separability of cell populations described by features measured in low resolution slit-scanning flow systems with their separability when the features are extracted from high resolution digitized cell images. The results show that although the accuracy of the feature measurements deteriorates for increasing slit width, this is not necessarily true for the discriminatory power of the features. Depending on their original position in the high resolution feature space, the cell populations may be located even farther apart in the space of low resolution slit-scan features for reasonably small widths of the slit. The results presented with high resolution images of cells from gynecological specimens and simulated slit-scan measurements can be explained by the model. For the features nuclear DNA content and diameter the abnormal populations are shifted closer to the normal populations in the slit-scan simulations as compared to the high resolution measurements. The cell classifier errors rates are unacceptably high. 相似文献
10.
The high-speed sampling requirements of multidimensional slit-scan signals (cell contours) have typically required custom hardware. This specialized hardware has often lacked the flexibility to adapt to varying instrument setups and experimental requirements. A hardware and software system capable of sampling multiple slit-scan cell contours at rates of up to 40 MHz with 10-bit resolution is described. It utilizes commercially available CAMAC transient recorders, a Digital Equipment Corp. PDP-11/83 computer, and custom hardware for signal conditioning and trigger generation. The modular design of the software system allows various hardware options with minimal additional coding. Real-time digital processing checks each cell contour for multiple peaks; extracts morphological features such as width, height, and area; accumulates gated histograms of these data; and optionally saves the derived data, selected contours, or both into list mode files on disk. 相似文献
11.
A multidimensional slit-scan flow system has been developed to serve as an automated prescreening instrument for gynecological cytology. Specimens are classified abnormal based on the number of cells having elevated nuclear fluorescence (alarms). An alarm region in a bivariate histogram of nuclear fluorescence versus nuclear-to-cell-diameter ratio is defined. Alarm region probability arrays are calculated to estimate the probability that an alarm falling in a particular bin of the alarm region is either from a normal or an abnormal specimen. From these arrays, a weighted alarm index is generated. In addition, summary indices are derived that measure how the distribution of alarms in each specimen compares with the average distributions for the normal and abnormal specimen populations. These indices together with current features are evaluated with respect to their utility in specimen classification using a nonparametric classification technique known as recursive partitioning. Resulting classification trees are presented that suggest information in the distribution of alarms in the bivariate histogram. In addition, they validate the features and rules currently used for specimen classification. Recursive partitioning appears to be useful for multivariate classification and is seen as a promising technique for other applications. 相似文献
12.
Antibodies have been prepared against the three major polypeptides of the nuclear pore complex-lamina fraction from rat liver nuclei. The three antisera prepared in chickens give similar results in indirect immunofluorescence microscopy. In rat embryo fibroblasts we observe bright fluorescence at the level of the nuclear envelope, with no fluorescence of the nuclear interior and little or no fluorescence of the cytoplasm. The nuclear envelope regions of rat hepatoma cells, mouse A9 cells, HeLa cells and rat liver nuclei also fluoresce brightly. HeLa nucleoids, which are depleted of nuclear envelope components, still exhibit specific fluorescence when reacted with these antibodies. Distribution of the antigens changes during mitosis. Fluorescence in the cytoplasm is observed following the breakdown of the nuclear envelope at prometaphase. The antigens appear to progressively accumulate at the periphery of the chromosomes until telophase. In late telophase fluorescence occurs predominantly at the periphery of the chromosomes where the new nuclear envelope is formed. 相似文献
13.
We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit-scan flow cytometry. Fluorescence scans of R-banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R-banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1-12. Scans obtained for chromosomes 13-22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R-bands by slit-scan flow cytometry. Specific patterns were detected for human chromosomes 9-12 in which the number and placement of the fluorescent bands served as classifiers. 相似文献
14.
E T Mystkowska A Komar P Strojny M Rozycka W Sawicki 《Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology》1991,13(3):209-214
Living two-cell mouse embryos were flushed out from the oviduct 17, 24 and 36 hours after fertilization in order to obtain cells in the G1S, early G2 and late G2 phases of the second cell cycle. The nuclei of the living cells were stained with Hoechst 33342. The coordinates of the contour shapes of the entire cells (cellular contours) were registered by contour image processing with a TV camera coupled with a computer; the contours of the nuclei were computed by means of a digitizer coupled with the computer. Fourier analysis of the cellular and nuclear contours revealed systematic modifications in the folding of the cells and nuclei in the course of the murine second cell cycle. The progression of cells through the second cell cycle was correlated with an increasing diversification of cellular and nuclear shape, with the diversification being much more pronounced in the nuclear shapes. 相似文献
15.
We conducted high-resolution fluorescent image analysis with mithramycin-stained cells from clinical gynecologic specimens. Features characteristic of the usual, low-resolution, one-dimensional, slit-scan flow cytometric measurements were extracted from 250 high-resolution nuclear images. In addition to the measurement of the usual parameters, nuclear ellipticity and DNA density (DNA per unit nuclear size) were also determined. Our preliminary results indicate that both of these features offer increased discrimination. When nuclear shape was included as a global feature, at least 77% of the diagnostic cells could be distinguished from normal cells, with no overlap. Both features hold promise for improving the discrimination possible with flow cytometry. 相似文献
16.
J W Gray D Peters J T Merrill R Martin M A Van Dilla 《The journal of histochemistry and cytochemistry》1979,27(1):441-444
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. 相似文献
17.
Light scatter from epithelial cells in a slit-scan flow system is modeled using the Fraunhofer condition of scalar diffraction theory. Power spectra are calculated for successive positions of model cells in the line focus of a laser beam with a Fourier transform computer program. Using the calculated power spectra, detector configurations are designed to detect specific cell structures of interest. Detector configurations are tested in a static slit-scan scatter apparatus. Data indicating the ability to detect boundaries and cell orientation are discussed. 相似文献
18.
J A Steinkamp K M Hansen H A Crissman 《The journal of histochemistry and cytochemistry》1976,24(1):292-297
A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented. 相似文献
19.
Sorting on the basis of the complex features resolved by chromosome slit-scan analysis requires rapid and flexible pulse shape acquisition and processing for determining sort decisions before droplet breakoff. Fluorescence scans of chromosome morphology contain centromeric index and banding information suitable for chromosome classification, but these scans are often characterized by variability in length and height and require sophisticated data processing procedures for identification. Setting sort criteria on such complex morphological data requires digitization and subsequent computation by an algorithm tolerant of variations in overall pulse shape. We demonstrate here the capability to sort individual chromosomes based on their morphological features measured by slit-scan flow cytometry. To do this we have constructed a sort controller capable of acquiring an 128 byte chromosome waveform and executing a series of numerical computations resulting in an area-based centromeric index sort decision in less than 2 ms. The system is configured in a NOVIX microprocessor, programmed in FORTH, and interfaced to a slit-scan flow cytometer data acquisition system. An advantage of this configuration is direct control over the machine state during program execution for minimal processing time. Examples of flow sorted chromosomes are shown with their corresponding fluorescence pulse shapes. 相似文献
20.
A simple circuit is described for generating a variety of electronic pulses to test hardware and software for slit-scan chromosome analysis in a flow cytometer. The pulse shape can be changed to have different numbers of local minima, thereby simulating fluorescence pulses from acrocentric, monocentric, and dicentric chromosomes. Long pulses simulate aggregates of chromosomes. The pulse repetition rate as well as the pulse amplitude is variable. Although the circuitry is built with only three integrated circuits, the pulse-to-pulse variation in shape and height is quite small. After digitization of the analog signals, the constructed histograms of pulse integrals show a relative coefficient of variation below 1%. This signal generator provides a valuable tool for a number of electronic test applications that would otherwise require expensive standard particles analyzed in a well-tuned flow cytometer. 相似文献