Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

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.     

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.     

An optical platform for cell tracking in adult zebrafish

Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.     

Characterization of bacteria by multiparameter flow cytometry

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

Characterization of bacteria by multiparameter flow cytometry.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

Flow cytometry: instrumentation and application in phytoplankton research

In flow cytometry, light scattering and fluorescence of individual particles in suspension is measured at high speed. When applied to planktonic particles, the light scattering and (auto-)fluorescence properties of algal cells can be used for cell identification and counting. Analysis of the wide size spectrum of phytoplankton species, generally present in eutrophic inland and coastal waters, requires flow cytometers specially designed for this purpose. This paper compares the performance in phytoplankton research of a commercial flow cytometer to a purpose built instrument. It reports on the identification of phytoplankton and indicates an area where flow cytometry may supersede more conventional techniques: the analysis of morphological and physiological characteristics of subpopulations in phytoplankton samples.     

Orientation measurements of microsphere doublets and metaphase chromosomes in flow

Obtaining information about the shape of particles from slit-scan profiles is facilitated if the particles are oriented. Elongated particles orient in the nozzle of flow cytometers, but orientation may be disrupted before the particles get to the point of measurement. We have used our slit-scan flow cytometer to investigate the orientation of microsphere doublets in a liquid jet in air, in flow across a glass surface, and in a 200-microns-square capillary tube as a function of distance from the flow chamber nozzle. Particles were classified as being oriented if there was a centrally located dip in the slit-scan profile. Essentially all the doublets in the jet were oriented, and no disorientation was noted over the distances measured (up to 10 mm from the nozzle). Particle orientation was maintained for 80 microns in flow across a glass surface. In the capillary-type flow chamber, essentially all of the particles were oriented at the tube entrance and for several millimeters into the tube. There then occurred a region where particle tumbling started and progressively fewer doublets met the orientation criteria. The distance to where tumbling began could be estimated by calculating the length required to establish the parabolic flow profile in the tube. Finally, the fraction of oriented particles reached a constant value that did not change with increased distance into the tube. When sample was injected off axis (i.e., halfway between the chamber center and the chamber walls), particle tumbling began closer to the tube entrance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection

Optical waveguides represent alternatives to lenses for delivery of light to, and collection of light from, small regions of space such as the observation point(s) of a flow cytometer. The flow chamber and directly associated illumination and collection optics of a flow cytometer can be built using fiber optic waveguides in fixed positions or as an integrated optical assembly incorporating deposited or implanted waveguides, providing illumination and collection geometries and efficiencies comparable to conventional designs, with the advantage that the optical waveguide design, once assembled, never again requires alignment of the optics. A prototype apparatus in which fiber optics were used to illuminate and collect light from a point inside a thin-walled, round quartz capillary was observed to measure fluorescence of polystyrene spheres with precision close to that obtained from a conventional flow cytometer using the same laser source. The design of the fiber optic system is readily adaptable to construction of multiple illumination beam instruments; other optical waveguide flow cytometer designs that dispense with the capillary share this advantage and can also provide orifices for electronic volume sensing and for jetting prior to droplet sorting.     

The ImageStream System: a key step to a new era in imaging

The aim of this article is to provide a brief review about the ImageStream system a novel tool for multiparameter cell analysis in flow. The instrument integrates the features of flow cytometry and fluorescence microscopy combined with a modern methodology for image analysis. Similar to flow cytometry, ImageStream allows analysis of a large number of cells based on their fluorescence features and provides statistical analysis of these features. Additionally, ImageStream allows detailed morphometric cellular analysis based on acquired cellular images integrating various morphometric and photometric features of the examined cells. Simply stated, ImageStream system is an advanced flow cytometer acquiring both integrated fluorescence signals as well as high quality fluorescence images and allowing muliparameter analysis. The innovative features of the instrument offer new analytical capabilities and allow for a multitude of possible applications beyond the current means of flow cytometry. While this article summarizes basic information about the features of ImageStream and its applications based on the available literature and it also describes our own experience.     

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.     

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology

BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.     

Simultaneous separate detection of low angle and large angle light scattering in an arc lamp-based flow cytometer

A device is described for simultaneous separate detection of the light scattering of cells at low and large scattering angles in an arc lamp-based flow cytometer with epi-illumination through an oil immersion microscope objective. Light scattering was measured in a dark field configuration that allows separate detection of light scattering greater than 2 degrees and 15 degrees, respectively. Dual parameter light scattering histograms of a blood cell suspension containing various types of leukocytes were closely similar to that obtained with a commercial laser-based instrument with light scattering detection at forward and right angles. The sensitivity of the device was sufficient to measure polystyrene particles with 0.25-micron diameter. A potential application may be differentiation of bacteria.     

Detection and discrimination of individual viruses by flow cytometry.

A new flow cytometer with a very small observation volume has been developed to detect individual viruses with good resolution, and has been used to discriminate between two types of viral particles based on differences in their light scattering. Measurements of light scattering and fluorescence made with such an instrument can provide a basis for quantitative analysis and sorting of viruses and other particles in the micron and submicron size range.     

Practical time-gated luminescence flow cytometry. II: experimental evaluation using UV LED excitation.

In the previous article [Part 1 (8)], we have modelled alternative approaches to design of practical time-gated luminescence (TGL) flow cytometry and examined the feasibility of employing a UV LED as the excitation source for the gated detection of europium dye labelled target in rapid flow stream. The continuous flow-section approach is well suited for rare-event cell counting in applications with a large number of nontarget autofluorescent particles. This article presents details of construction, operation and evaluation of a TGL flow cytometer using a UV LED excitation and a gated high-gain channel photomultiplier tube (CPMT) for detection. The compact prototype TGL flow cytometer was constructed and optimised to operate at a TGL cycle rate of 6 kHz, with each cycle consisting of 100 micros LED pulsed excitation and approximately 60 micros delay-gated detection. The performance of the TGL flow cytometer was evaluated by enumerating 5.7 microm Eu(3+) luminescence beads (having comparable intensity to europium-chelate-labeled Giardia cysts) in both autofluorescence-rich environmental water concentrates and Sulforhodamine 101 (S101) solutions (broadband red fluorescence covering the spectral band of target signals), respectively.The prototype TGL flow cytometer was able to distinguish the target beads, and a maximum signal to background ratio of 38:1 was observed. Neither the environmental water concentrates nor S101 solution contributed to the background in the TGL detection phase. The counting efficiency of the TGL flow cytometer was typically >93% of values determined using conventional counting methods.     

流式细胞术在细菌快速检测中的应用

流式细胞仪(Flow cytometer)是集应用流体学、光学、电子学、生物学、免疫学等多门学科和技术于一体的新型高科技仪器。它的核心技术是流式细胞术(Flow cytometry,FCM),该技术是利用流式细胞仪,使单个细胞或其他微小生物粒子处于快速直线流动状态,且逐个通过光束,从而对单个细胞或微粒进行多参数(数量、大小、核酸含量、细胞活性、特定菌群或物种等)定量分析和分选的检测技术,具有快速、灵敏、精确以及便于操作等突出优点。本文简要介绍流式细胞仪的原理,并论述流式细胞技术在实验室研究、工业生产、临床诊断、环境评估等领域的细菌快速检测应用。     

Optical plankton analyser: a flow cytometer for plankton analysis, II: Specifications

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton analysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 microns in width and over 1,000 microns in length can be analysed. Sample flow rates of up to 55 microliters/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 microns. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 10(6). A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.     

Design and characterization of a compact dual channel virus counter.

BACKGROUND: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. METHODS: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. RESULTS: The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. CONCLUSIONS: The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).     

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.     

Hyperspectral cytometry at the single-cell level using a 32-channel photodetector

Despite recent progress in cell-analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate-up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8-15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well-separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next-generation flow cytometer based on a 32-channel PMT array detector, a phase-volume holographic grating, and a fast electronic board. The system is capable of full-spectral data collection and spectral analysis at the single-cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in <5 μs. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data-collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification.