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.     

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.     

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.     

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.     

Imaging systems for correlation of false alarms in flow.

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.     

Software Architecture for Processing Clusters Based on I2O

Mainstream computing equipment and the advent of affordable multi-Gigabit communication technology permit us to address data acquisition and processing problems with clusters of COTS machinery. Such networks typically contain heterogeneous platforms, real-time partitions and even custom devices. Vital overall system requirements are high efficiency and flexibility. In preceding projects we experienced the difficulties to meet both requirements at once. Intelligent I/O (I₂O) is an industry specification that defines a uniform messaging format and execution environment for hardware and operating system independent device drivers in systems with processor based communication equipment. Mapping this concept to a distributed computing environment and encapsulating the details of the specification into an application-programming framework allow us to provide architectural support for (i) efficient and (ii) extensible cluster operation. This paper portrays our view of applying I₂O to high-performance clusters. We demonstrate the feasibility of this approach and report on the efficiency of our XDAQ software framework for distributed data acquisition systems.     

Preselection of alarms in a hybrid system for screening of cervical cancer.

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.     

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.     

ScanImage: Flexible software for operating laser scanning microscopes

Background  

Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope.     

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.     

A computer system for real-time data acquisition and analysis of biopotentials and quantal content at the neuromuscular junction

A computerized data acquisition system for on-line analysis of the parameters of neuromuscular transmission is described. Both hardware usage and software methodologies are discussed with regard to sampling in real-time and analyzing miniature end-plate potentials (MEPPs), end-plate potentials (EPPs) and quantal content of the evoked transmitter release. Significant features of the program include: (1) automatic threshold determination for MEPP detection; (2) the use of a circular buffer to give pre-trigger information; (3) real-time noise spike rejection; (4) an automatic procedure for EPP failure detection; (5) rapid quantal content determinations by several methods as well as complete MEPP and EPP waveform analysis. The system has proven both accurate and reliable during more than two years of use. Advantages of the system over conventional methods include: (1) increased accuracy and efficiency in data analysis; (2) immediate availability of results; (3) conventional data storage; (4) flexibility to meet changing requirements.     

Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening

Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed‐batch processes, including at‐line sampling, deep freezer off‐line sample storage, and complete data handling. To enable scalable high‐throughput early‐stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom‐built hardware and software, fully automated milliliter‐scale microbial fed‐batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high‐throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early‐stage bioprocess development.     

Application of Fraunhofer diffraction theory to feature-specific detector design.

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.     

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.     

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.     

Cellular assay optimization: part II: the use of a simple integrated robotic work cell to allow the multiplexed batching of cellular assays

This report describes the implementation of an automated work cell with commercially available hardware and software, capable of handling up to 15 separate reagents for performing 96-well or 384-well assays but with a small footprint and only a single liquid dispenser and two plate washers. Extremely flexible software was used to enable this simple work cell to perform processes that would traditionally require a much larger, more expensive automation platform. With the development of the C-Myc assays for the targets DYRK, BMX, PERK, and FAK, the authors describe a software solution to multibatch assays to run simultaneously, reducing reagent dead volume and increasing the efficiency of running multiple assays such that the time to generate data across multiple targets was significantly shortened. Although a larger automated system with multiple robotic arms and extensive equipment would also be able to process multiple assays simultaneously, the work cell we have described represents an inexpensive and flexible, easily upgradable option suitable for a wider range of labs.     

基于MIMIC数据库的多参数数据D/A回放系统

文中分析了MIMIC多参数智能监护数据库存储文件的识读方法,该数据库公开发表于PhysioNET,还研究及基于STM32和DAC8568的多参数数据D/A回放系统,包括系统硬件工作原理和系统软件工作原理。MIMIC多参数智能监护数据库的数据文件对于变异性较大的生理参数采用连续采样的方式,而对于相对稳定的生理参数采用周期采样的方式,因此,多参数数据各信号的抽样频率不同。系统首先通过VC++编程读取一个多抽样频率数据并在PC机上显示其波形,然后经过D/A硬件设备进行D/A转换后使用示波器输出其波形,实现输出的波形与原始的波形能同时回放。系统可作为模拟信号源,为远程多参数生理监护仪的研制奠定了基础。     

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.     

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 pulse generator simulating slit-scan chromosome analysis signals

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.