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。     

Current and future applications of flow cytometry in aquatic microbiology

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.     

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。     

Measurements of reflectance and fluorescence spectra for nondestructive characterizing ripeness of grapevine berries

In vivo reflectance and fluorescence spectra from berry skins of a white (Riesling) and red (Cabernet Sauvignon) grapevine variety were measured during a ripening season with a new CMOS radiometer instrument. Classical reference measurements were also carried out for a sugar content of the berry juice [°Brix] and pigment contents (chlorophyll a and b, carotenoids, anthocyanins) from methanol extracts of the berry skin. We showed that the colours and the spectra analysed from them could be taken as an unambiguous indicator of grapevine ripening. Reflectance spectra, which were affected by the content of pigments (chlorophylls and anthocyanins), effects of surface (wax layers), and tissue structure (cell size) of the berries well correlated (R² = 0.89) with the °Brix measurements of the berries. The fast data acquisition of both reflectance and fluorescence spectra in one sample with our radiometer instrument made it superior over the time-consuming, traditional, and mostly destructive chemical analysis used in vine-growing management.     

Flow cytometric analysis of fine particles in a eutrophic lake.

Fine particles play an important role, not only in aquatic biogeochemical processing but also in the distribution, transfer and transformation of pollutants in the aquatic environment. Flow cytometry, widely used in biomedical research, allows fast counting and optical analysis of individual particles. Organic autotrophic particles contain naturally fluorescing pigments, such as chlorophyll and phycoerythrin. Different populations have different sizes and pigments. They also have different ratios of pigments. In general, side angle scatter (SSC) is related to the size, shape and refractive index of particles. When a 488 nm wavelength was used to excite chlorophyll and phycoerythrin fluorescence, the pigments of organic autotrophic particles emitted red and orange light. Fine particles were detected by flow cytometry (FCM) in the southern part of a eutrophic lake in winter. We found that organic autotrophic particles belonged to three populations, which represented only 15.89% of total fine particles. Organic non-living particles and inorganic particles represented the greater part (84.11%) of total fine particles. This study also demonstrated that flow cytometry is well suited to the dynamic monitoring and analysis of natural water aquatic particles that were difficult to study with traditional methods.     

The use of isochronal reference standards in phase and modulation fluorescence lifetime measurements

Errors in phase and modulation lifetime measurements observed with the only commercially available instrument are readily apparent when the Debye-Sears modulation tank is not perfectly tuned. Unfortunately, we have found that exact tuning was extremely difficult to achieve and maintain. We report that these errors could be reduced by using single-lifetime (homogeneous) reference standards whose fluorescence lifetime approximated that of the unknown sample (isochronal standards). A number of useful standards are suggested. In the proposed method, the phase shift and relative modulation of the sample emission are measured using the isochronal standard as a reference to determine the effective characteristics of the sinusoidal excitation. The importance of the improvement in accuracy accomplished by the proposed methods is illustrated by the accurate resolution of fluorescence lifetime heterogeneity for two known heterogeneous samples.     

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。     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Commerical reference shape standards use in the study of particle shape effect on laser diffraction particle size analysis

The purpose of this paper is to describe the use of LGC Promochem AEA 1001 to AEA 1003 monosized fiberanalog shape standards in the study of the effect of particle shape on laser diffraction (LD) particle size analysis (psa). The psa of the AEA standards was conducted using LD psa systems from Beckman Coulter, Horiba, and Malvern Instruments. Flow speed settings, sample refractive index values, and sample cell types were varied to examine the extent to which the shape effect on LD psa results is modified by these variables. The volume and number probability plots resulting from these measurements were each characterized by a spread in the particle size distribution that roughly extended from the breadth to the longest dimension of the particles. For most of the selected sample refractive index values, the volume probability plots were characterized by apparent bimodal distributions. The results, therefore, provide experimental verification of the conclusions from theoretical studies of LD psa system response to monosized elliptical particles in which this apparent bimodality was the predicted result in the case of flow-oriented particles. The data support the findings from previous studies conducted over the past 10 years that have called into question the verity of the tenets of, and therefore the value of the application of, the equivalent spherical volume diameter theory and the random particle orientation model to the interpretation of LD psa results from measurements made on nonspherical particles.     

Commercial reference shape standards use in the study of particle shape effect on laser diffraction particle size analysis

The purpose of this paper is to describe the use of LGC Promochem AEA 1001 to AEA 1003 monosized fiber-analog shape standards in the study of the effect of particle shape on laser diffraction (LD) particle size analysis (psa). The psa of the AEA standards was conducted using LD psa systems from Beckman Coulter, Horiba, and Malvern Instruments. Flow speed settings, sample refractive index values, and sample cell types were varied to examine the extent to which the shape effect on LD psa results is modified by these variables. The volume and number probability plots resulting from these measurements were each characterized by a spread in the particle size distribution that roughly extended from the breadth to the longest dimension of the particles. For most of the selected sample refractive index values, the volume probability plots were characterized by apparent bimodal distributions. The results, therefore, provide experimental verification of the conclusions from theoretical studies of LD psa system response to monosized elliptical particles in which this apparent bimodality was the predicted result in the case of flow-oriented particles. The data support the findings from previous studies conducted over the past 10 years that have called into question the verity of the tenets of, and therefore the value of the application of, the equivalent spherical volume diameter theory and the random particle orientation model to the interpretation of LD psa results from measurements made on nonspherical particles.     

Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the "best primary standards"

Flow cytometry (FCM) techniques have enabled characterization of the genome size for various plant species. In order to measure the nuclear genome size of a species, reference standards with well-established DNA content are necessary. However, different 2C-values have been described for the same species used as reference standard. This fact has brought about inaccurate genome measurements, making relevant the establishment of optimal DNA reference standards for plant cytometric analyses. Our work revisited the genome size of Arabidopsis thaliana and other seven plant standards, which were denominated ??Dole?el??s standard set?? and have been widely used in plant DNA measurements. These eight plant standards were reassessed for a comparative measurement of their DNA content values, using each plant species as primary standard in a cascade-like manner, from A. thaliana to Allium cepa. The genome size values obtained here were compared to those reported in the literature by statistical analyses. As a result, Raphanus sativus and Drosophila melanogaster were considered the most inadequate primary standards, whereas A. thaliana, Solanum lycopersicum and Pisum sativum were found to be the most suitable.     

Use of a neural net computer system for analysis of flow cytometric data of phytoplankton populations

Flow cytometry has been used over the past 5 years to begin detailed exploration of the distribution and abundance of picoplankton in the oceans. Light scattering and fluorescence measurements on individual plankton cells in seawater samples allow construction of population signatures from size and pigment characteristics. The use of "list mode" data has made these studies possible, but on-shore analysis of copious data does not permit on-site reexamination of important or unexpected observations, and overall effort is greatly handicapped by data analysis time. Here we describe the application of neural net computer technology to the analysis of flow cytometry data. Although the data used in this study are from oceanographic research, the results are general and should be directly applicable to flow cytometry data of any sort. Neural net computers are ideally suited to perform the pattern recognition required for the quantitative analysis of flow cytometry data. Rather than being programmed to perform analysis, the neural net computer is "taught" how to analyze the cell populations by presenting examples of inputs and correct results. Once the system is "trained," similar data sets can be analyzed rapidly and objectively, minimizing the need for laborious user interaction. The neural network described here offers the advantages of 1) adaptability to changing conditions and 2) potential real-time analysis. High accuracy and processing speed near that required for real-time classification have been achieved in a software simulation of the neural network on a Macintosh SE personal computer.     

Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement

BACKGROUND: Telomeres are highly conserved repeats at the ends of chromosomes that maintain chromosome stability and reflect the replicative potential of cells. Telomere length can be determined by Southern blot hybridization or quantitative fluorescence in situ hybridization (Q-FISH). Recently, two flow cytometry-based (Flow) FISH protocols have been published. METHODS: We compared the telomere length measured by Southern blotting and Flow FISH using standard beads to calibrate and quantify the fluorescence intensity. RESULTS: The telomeric fluorescence of cord blood and peripheral blood mononuclear cells was similar to that reported by other studies. There was a linear relationship between the telomeric fluorescence determined by Flow FISH and the telomere fragment size determined by Southern blotting (r = 0.89; P < 0.001). CONCLUSION: It is important to set up a center-specific curve and select appropriate cell lines for reference. This Q-Flow FISH protocol will facilitate the measurement of telomere length and allow more meaningful comparison of data (in standard fluorescence units or fragment size) between institutes.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Optimizing optical flow cytometry for cell volume-based sorting and analysis

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work.     

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.     

Characterization of intracellular accumulation of poly-beta-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry

Poly-beta-hydroxybutyrate (PHB) accumulates in individual cells of Alcaligenes eutrophus in the form of refractile bodies which alter the light-scattering properties of individual cells. Flow cytometry has been applied to measure the distributions of single-cell light-scattering intensity in Alc. eutrophus populations during batch cultivation of the organism. These measurements clearly identify heterogeneities in the inoculum which influence the lag interval prior to beginning of exponential growth. Light-scattering distributions show greater homogeneity and are extremely similar during balanced, exponential growth. After exhaustion of the nitrogen source and with carbon source still available, significant PHB accumulations occur and the flow cytometry measurements reveal extreme heterogeneity in single-cell light-scattering properties. These measurements clearly demonstrate the potential advantages of single-cell light-scattering measurements by flow cytometry for analysis and control of certain fermentation processes. Single-cell light-scat light-scattering measurements in conjunction with flow sorting instrumentation have been applied to demonstrate enrichment of PHB-producing cells, initially present in a number concentration of 0.01%by a factor of 300 in a single pass. Flow cytometry-cell sorting technology should find significant application in strain improvement and mutant selection.     

A simple electronic volume cell sorter for clonogenicity assays

A single-parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to approximately 2,000 per second with greater than 80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including 1) precise cell plating for low-dose survival studies, 2) separation of cells into age compartments, and 3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.     

Flow cytometry applications in the food industry

Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.     

Flow cytometry of human gynecologic specimens using log chromomycin A3 fluorescence and log 90 degrees light scatter.

Flow cytometry and electronic cell sorting are being investigated to screen gynecologic specimens for cervical neoplasia. Cellular DNA content is quantitated by Chromomycin A3 fluorescence and cell size is quantitated by 90 degrees light scatter; the logarithms of the measured intensities are used to produce a two parameter histogram. To determine the cell types responsible for signals in various histogram regions, systematic electronic cell sorting is performed. The sorted fractions are sedimented into microscope slides and stained by the Papanicolaou technique. The cells in each fraction are identified by conventional cytomorphologic criteria. Morphologic analysis of sorted cells reveals histogram regions corresponding to specific cell types. One very important region contains the highest concentration of signals from abnormal cells and is therefore the best region to analyze for specimen abnormality. However, because a significant number of signals in this region are from normal cells, specimens cannot be diagnosed by their analysis. Another important histogram region is composed primarily of signals from endocervical columnar and metaplastic cells. The presence of such cells is a good criterion for specimen adequacy, therefore analysis of signals in this region is essential to assess specimen adequacy for automatic screening.