Optical plankton analyser: a flow cytometer for plankton analysis, I: Design considerations

The design criteria for a flow cytometer (FCM) for the analysis of field samples of phytoplankton are described. The criteria are based on the occurrence of a wide variety of particle sizes in field samples, normally at low concentrations. The instrument should be able to analyse cells and colonies from 0.5 to 500 microns diameter and of over 2,000 microns length. A minimum flow rate of 4 microliters.s-1 was calculated from natural plankton concentrations. Commercially available FCMs are not suited to measure this range of sizes at this rate. Further limitations of standard FCMs are uneven illumination or incomplete processing of long signals. In addition, long filamentous colonies can break into small fragments caused by too high acceleration in the standard flow cuvette. Recognition of these limitations is of importance for the flow cytometry of phytoplankton. The new design was developed to avoid these limitations. A dynamic range 5 to 6 decades could be accomplished by a combination of logarithmic amplifiers, a slit-shaped focal spot, and a pulse integration system that can process long pulses. Multilaser capability to identify different phytoplankton species, a low fluid shear cuvette, and a trigger gate-extension for inhomogeneously fluorescent algal filaments were included in the design.     

A low-cost, portable flow cytometer specifically designed for phytoplankton analysis

This paper describes the construction and testing of a flowcytometer illuminated by a low-power (40 mW), air-cooled argonlaser and incorporating a novel flow cell with a wide (0.3 mm)nozzle for hydrodynamic focusing. The resolution of the instrumentis comparable to that of commercial equipment operating at muchhigher laser powers, and it is sensitive enough to detect autofluorescencefrom single picoplankton cells. By using recent innovationsin laser technology and electronics, the design allows a research-qualityflow cytometer to be constructed at relatively low cost ({smalltilde}£15 000 at current prices). The instrument doesnot require cooling water, three-phase power or compressed gassupplies, and is easily portable from one operating site toanother. The large focusing nozzle is very resistant to cloggingby detritus, cell clumps or filaments, but is still capableof producing cores down to 5 µm diameter. These characteristicsare particularly suitable for the analysis of microalgal culturesand phytoplankton samples. The discriminatory power of the instrumenthas been tested using a range of marine and freshwater algae.     

Modification of a laser-based flow cytometer for high-resolution DNA analysis of mammalian spermatozoa

Modification of a Coulter EPICS V orthogonal laser-based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome-bearing spermatozoa from the bull, boar, and ram can be resolved using this system.     

Ultrasonic particle-concentration for sheathless focusing of particles for analysis in a flow cytometer.

BACKGROUND: The development of inexpensive small flow cytometers is recognized as an important goal for many applications ranging from medical uses in developing countries for disease diagnosis to use as an analytical platform in support of homeland defense. Although hydrodynamic focusing is highly effective at particle positioning, the use of sheath fluid increases assay cost and reduces instrument utility for field and autonomous remote operations. METHODS: This work presents the creation of a novel flow cell that uses ultrasonic acoustic energy to focus small particles to the center of a flowing stream for analysis by flow cytometry. Experiments using this flow cell are described wherein its efficacy is evaluated under flow cytometric conditions with fluorescent microspheres. RESULTS: Preliminary laboratory experiments demonstrate acoustic focusing of flowing 10-microm latex particles into a tight sample stream that is approximately 40 microm in diameter. Prototype flow cytometer measurements using an acoustic-focusing flow chamber demonstrated focusing of a microsphere sample to a central stream approximately 40 microm in diameter, yielding a definite fluorescence peak for the microspheres as compared with a broad distribution for unfocused microspheres. CONCLUSIONS: The flow cell developed here uses acoustic focusing, which inherently concentrates the sample particles to the center of the sample stream. This method could eliminate the need for sheath fluid, and will enable increased interrogation times for enhanced sensitivity, while maintaining high particle-analysis rates. The concentration effect will also enable the analysis of extremely dilute samples on the order of several particles per liter, at analysis rates of a few particles per second. Such features offer the possibility of a truly versatile low-cost portable flow cytometer for field applications.     

Nine fluorescence parameter analysis on a four-color fluorescence activated flow cytometer.

BACKGROUND: In many cases a frequent monitoring of blood is important, e.g. during an infection. Often the availability of blood is the limiting factor. METHODS: 50 microl blood were stained and analyzed using a standard four-color cytometer. Percentages of leukocytes were calculated by FlowJo software. RESULTS: Our protocol allows the differentiation of B cells, T cells, NK cells, neutrophils, and monocytes/macrophages in small volumes of blood. CONCLUSIONS: Using nine fluorochrome-labeled antibodies and a specific gating strategy we were able to differentiate the main immune cells in minute amounts using one staining step.     

Characteristics of a simple, high-resolution flow cytometer based o a new flow configuration.

A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.     

Calibration of a flow cytometer against a microphotometer for morphologic cell identification

The calibration of a flow cytometer against a microphotometer, to allow the correlation of cell morphology with fluorescence intensity, is described. Using three human lymphoblastoid cell lines, the photomultiplier amplification of the microphotometer and the flow cytometer that gave optimum linearity between fluorescence intensity and DNA content for the two instruments was established. Thereafter, at these settings, there was satisfactory linear agreement between the fluorescence intensity profiles, as measured by the flow cytometer and the microphotometer, of stained cell populations. Day-to-day variation was also minimal, and it was demonstrated that the application of this procedure can provide an alternative to the employment of the sorting facility of a flow cytometer for the morphologic identification of cell subpopulations during flow cytometric analysis.     

Setting up and running a microscope-based flow cytometer

A noncommercial flow cytometry system was set up in a laboratory with no engineering staff attached. The system is based upon a conventional fluorescence microscope and a new flow principle reported by H. Steen and T. Lindmo (Science 204:403, 1979). A few modifications and additions have been introduced. The instrument has proven to be a high-resolution system, easy to adjust and control, giving coefficients of variation of cellular DNA down to 0.8% and of fluorescent polyethylene microspheres down to 0.9%.     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒(如细胞,细菌)的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来:功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

Macintosh graphics for the EPICS flow cytometer user

Graphic options for the EPICS flow cytometer user have been restricted in the past to software written specifically for the analysis and graphics of flow cytometric data. The software is limited to only a few graphic presentation styles. The technique described will allow the EPICS user to translate histogram files into text files that can be used in an alternative computer format.     

A parallel microfluidic flow cytometer for high-content screening

A parallel microfluidic cytometer (PMC) uses a high-speed scanning photomultiplier-based detector to combine low-pixel-count, one-dimensional imaging with flow cytometry. The 384 parallel flow channels of the PMC decouple count rate from signal-to-noise ratio. Using six-pixel one-dimensional images, we investigated protein localization in a yeast model for human protein misfolding diseases and demonstrated the feasibility of a nuclear-translocation assay in Chinese hamster ovary (CHO) cells expressing an NFκB-EGFP reporter.     

Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis

The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.     

Display of three-parametric data acquired by a flow cytometer

A program for producing contour maps of three-parameter flow cytometric data is described. The program is written in Pascal and can be implemented on any microcomputer with a graphics screen.     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒（如细胞,细菌）的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来：功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.     

Integration of a barcode reader with a commercial flow cytometer.

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time. The system uses a standard "off-the-shelf" bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tube reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.     

Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.

BACKGROUND:The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. METHODS AND RESULTS: Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CONCLUSIONS:CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.     

Interfacing a flow cytometer computer with a PC-based patient information system

We have established an interface between our flow cytometer's computer and the personal computer (PC) which supports our patient database system. The PC has been equipped with a commercially available IEEE-488 bus interface board which is connected to the interface bus of the cytometer's Hewlett-Packard 9000/300 computer (HP). The PC is set as a bus device with the same address as that of the HP's printer. It is programmed to examine the stream of data sent to the printer and extract from it and store in an MS-DOS text file selected information which subsequently may be transferred to the database system.