Signal analysis of slit-scan contour data.

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.     

EMOSS: an epiillumination microscope objective slit-scan flow system

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.     

Technique for cellular fluorescence distribution analysis

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.     

A multidimensional slit-scan flow system.

A new slit-scan type flow system is described which provides three (X, Y, and Z) orthogonal one-dimensional projections of cell fluorescence. A photomultiplier tube and two semiconductor array detectors are used to obtain the three slit-scan contours from cells traversing a single fluorescence excitation beam. A high speed, dedicated preprocessor analyzes the three contours in parallel, extracting certain features useful for rejecting cells from which an accurate measurement of nuclear fluorescence cannot be obtain. Contour data is buffered and transferred to a PDP-11/40 computer where nuclear fluorescence is measured and cells are classified. It is anticipated that this new instrument will provide a significant reduction in false alarm rate when applied to prescreening of gynecologic cytology specimens.     

Slit-scan cytofluorometry. Basis for automated prescreening of urinary tract cytology.

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.     

Slit-scan flow cytometry: separability properties of cell features

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.     

A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

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.     

System for flow sorting chromosomes on the basis of pulse shape

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.     

An EPROM-based programmable contour generator for use in flow cytometry

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.     

Expectation values for low-resolution slit-scan flow prescreening: influence of nuclear shape and DNA density

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.     

Imaging in flow.

Imaging in flow has been valuable in investigating discrepancies in flow cell measurements due to cell orientation and flow dynamics. This paper discusses optical consideration in flow imaging, slit and full field imaging systems and various cell motion arresting techniques from the standpoint of image plane exposure and suitable detector choices. It concludes with an explanation of the slit-imaging techniques employed in a multidimensional slit-scan flow system and slit-scan correlation system.     

Prediction of cervical neoplasia diagnosis groups. Discriminant analysis on digitized cell images

The purpose of this study was to develop discriminant analysis models for predicting cervical dysplasia/neoplasia case diagnoses using cytometric features derived from the digital image analysis of cell monolayers. The data base consisted of 925 cells from 27 cases diagnosed either as moderate dysplasia (n = 10), severe dysplasia (n = 5), carcinoma in situ (n = 8) or invasive carcinoma (n = 4) on both tissue biopsy and monolayer preparations. Cell features examined were cell diameter, nuclear diameter, nuclear mean optical density (OD), nuclear integrated OD (IOD), nuclear OD standard deviation, normalized IOD, nuclear texture and nuclear-cytoplasmic ratio. Features derived from cells visually classified as moderate dysplasia correctly predicted the case diagnosis of moderate dysplasia versus more severe disease for 85% of the cells. Prediction models using summary measures (mean and variance) derived from all visually classified abnormal cells within each case correctly separated all cases into their respective diagnostic categories. These findings suggest that dysplastic cells in a cytologic sample have features that collectively reflect the tissue diagnosis, regardless of the visual differences among the cells. Such information has potential use for diagnosis and possibly for prognosis.     

Applicability of Preoperative Nuclear Morphometry to Evaluating Risk for Cervical Lymph Node Metastasis in Oral Squamous Cell Carcinoma

Background

We previously reported the utility of preoperative nuclear morphometry for evaluating risk for cervical lymph node metastases in tongue squamous cell carcinoma. The risk for lymph node metastasis in oral squamous cell carcinoma, however, is known to differ depending on the anatomical site of the primary tumor, such as the tongue, gingiva, mouth floor, and buccal mucosa. In this study, we evaluated the applicability of this morphometric technique to evaluating the risk for cervical lymph node metastasis in oral squamous cell carcinoma.

Methods

A digital image system was used to measure the mean nuclear area, mean nuclear perimeter, nuclear circular rate, ratio of nuclear length to width (aspect ratio), and nuclear area coefficient of variation (NACV). Relationships between these parameters and nodal status were evaluated by t-test and logistic regression analysis.

Results

Eighty-eight cases of squamous cell carcinoma (52 of the tongue, 25 of the gingiva, 4 of the buccal mucosa, and 7 of the mouth floor) were included: 46 with positive node classification and 42 with negative node classification. Nuclear area and perimeter were significantly larger in node-positive cases than in node-negative cases; however, there were no significant differences in circular rate, aspect ratio, or NACV. We derived two risk models based on the results of multivariate analysis: Model 1, which identified age and mean nuclear area and Model 2, which identified age and mean nuclear perimeter. It should be noted that primary tumor site was not associated the pN-positive status. There were no significant differences in pathological nodal status by aspect ratio, NACV, or primary tumor site.

Conclusion

Our method of preoperative nuclear morphometry may contribute valuable information to evaluations of the risk for lymph node metastasis in oral squamous cell carcinoma.     

Assessment of ocular irritation by image processed quantification of cell injury in human corneal cell cultures and in corneal constructs

Currently, there are no accepted alternative tests for the replacement of animals in ocular irritation testing. This study focused on the quantification of cellular viability as a measure of toxic events in immortalised human corneal cell cultures and a three-dimensional corneal construct. Simultaneous vital dye staining by calcein AM and ethidium homodimer-1 was used to provide "live" and "dead" probes, respectively. For further quantification, we have developed image processing tools to evaluate digital images obtained from confocal fluorescence scanning microscopy measurements. Based on the finding that ocular irritation can be related to the extent of cell injury at the various cell layers of the cornea, we extended our studies from corneal cell cultures to an in vitro human corneal equivalent system comprising epithelial, stromal keratocyte and endothelial layers. Our results showed that the microscopic measurement of cellular injury by using either cell cultures or in vitro corneal constructs, combined with image processed quantification, can provide insight into the extent of the toxic effects.     

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).     

Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena.

Cellular events are accomplished by the coordinated interactions of cellular components within the three-dimensional context of a cell. Simultaneous observation of multiple components in three dimensions can be essential for understanding such interactions. Toward this end, we have developed a computerized microscope workstation capable of recording three-dimensional images of multiple cellular components in fixed and living cells. All aspects of microscope control, data collection, image processing and analysis can be performed on the one workstation. In this report, we describe the components and capabilities of this integrated system. In addition, we discuss some general problems of multiple-wavelength, three-dimensional imaging and our application of this technology to the analysis of chromosome organization in Drosophila melanogaster. Three-dimensional imaging of fixed embryos stained by indirect immunofluorescence has revealed the structural organization of chromosomes, microtubules, and the nuclear lamins. Imaging of living embryos injected with fluorescently labelled proteins has confirmed and extended these results by allowing the study of these structures throughout the cell cycle. The combination of the molecular specificity of fluorescence microscopy and the three-dimensional structural information obtained by our workstation has provided novel insights into the dynamic aspects of chromosome behavior during the cell cycle. We believe this system has many important applications in the study of the molecular basis of cellular events.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

Application of multiplexed cysteine‐labeled complex protein sample for 2D electrophoretic gel alignment

The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost‐effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co‐run with the sample. Maleimide‐conjugated fluorescent dyes Dy‐560 and Dy‐635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.     

阿达玛变换显微图象法测定乳腺癌DNA含量

以吖啶橙为细胞DNA荧光探针,用阿达玛(Hadamard)变换显微图象分析仪和显微荧光光度计分别测定了4例乳腺肿瘤的细胞DNA含量(倍性),并对分析结果进行了比较,二者的分析结果均与病理学诊断结论相吻合.阿达玛变换显微图象分析仪作为一种新的细胞定量分析仪器,其分析结果的准确度可与显微荧光光度计相媲美,而且阿达玛变换显微图象分析仪还具有其独特的优点,如信噪比高、具有同时分析多个细胞和同步扣除背景信号的能力等.