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.     

GenePattern flow cytometry suite

Background

Traditional flow cytometry data analysis is largely based on interactive and time consuming analysis of series two dimensional representations of up to 20 dimensional data. Recent technological advances have increased the amount of data generated by the technology and outpaced the development of data analysis approaches. While there are advanced tools available, including many R/BioConductor packages, these are only accessible programmatically and therefore out of reach for most experimentalists. GenePattern is a powerful genomic analysis platform with over 200 tools for analysis of gene expression, proteomics, and other data. A web-based interface provides easy access to these tools and allows the creation of automated analysis pipelines enabling reproducible research.

Results

In order to bring advanced flow cytometry data analysis tools to experimentalists without programmatic skills, we developed the GenePattern Flow Cytometry Suite. It contains 34 open source GenePattern flow cytometry modules covering methods from basic processing of flow cytometry standard (i.e., FCS) files to advanced algorithms for automated identification of cell populations, normalization and quality assessment. Internally, these modules leverage from functionality developed in R/BioConductor. Using the GenePattern web-based interface, they can be connected to build analytical pipelines.

Conclusions

GenePattern Flow Cytometry Suite brings advanced flow cytometry data analysis capabilities to users with minimal computer skills. Functionality previously available only to skilled bioinformaticians is now easily accessible from a web browser.

     

High throughput flow cytometry

BACKGROUND: Conventional flow cytometry does not allow the rapid analysis of multiple samples. This has limited its uses in drug discovery, for which the standard for throughput is 100,000 samples per day. METHODS: We describe a simple method in which commercial peristaltic tubing is connected from a commercial autosampler to a flow cytometer. The samples are delivered via a peristaltic pump from source wells in a multiwell plate. The samples are separated by air bubbles. RESULTS: Throughput rates approach the limit of the autosampler (up to 100 wells per minute). Using optimal tubing and flow rates, particles remain within appropriate light scatter and fluorescence gates. The carryover between wells is typically less than 5% without and 1% with a wash step. The volumes of sample delivered are in the microliter scale. The approach has been validated with instruments from three manufacturers. CONCLUSIONS: Flow cytometry has potential throughput of 100,000 samples or more per day starting with the method described. The method is currently best suited to end-point assays. However, combined with high-speed sorting and single- cell assays, the number of assays could approach 1 billion per day.     

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.     

Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances

Photoswitchable fluorescent proteins (PSFPs) with controllable spectral shifts in emission in response to light have led to breakthroughs in cell biology. Conventional photoswitching, however, is not applicable to weakly fluorescent proteins. As an alternative, photothermal (PT) and photoacoustic (PA) spectroscopy have demonstrated a tremendous potential for studying absorbing nonfluorescent proteins and nanoparticles. However, little progress has been made in the development of switchable PT and PA probes with controllable spectral shifts in absorption. Here, we introduce the concept of photothermally switchable nanoparticles (PTSNs). To prove the concept, we demonstrated fast, reversible magnetic–PT switching of conventional and gold‐coated magnetic nanoparticle clusters in cancer cells in vitro and PT switching of nonlinear ultrasharp plasmonic resonances in gold nanorods molecularly targeted to circulating cells in vivo. We showed that genetically encoded PSFPs with relatively slow switching can serve as triple‐modal fluorescent, PT, and PA probes under static conditions, while PTSNs with ultrafast switching may provide higher PA sensitivity in the near‐infrared window of tissue transparency under dynamic flow conditions. Application of nonlinear phenomena for super‐resolution spectral PT and PA cytometry, microscopy, and spectral burning beyond the diffraction and spectral limits are also proposed. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances

In the article by E. I. Galanzha et al. (doi: http://dx.doi.org/10.1002/jbio.201300140 ), published in J. Biophotonics 8, 81–93 (2015), the Conflict of Interest statement is missing. This erratum is published to correct this.     

Immunofluorescent flow cytometry inN dimensions

The problem of the simultaneous use in flow cytometry ofN>2 antibodies in conjunction with two fluorochromes was investigated. Theoretical analysis led to a labeling procedure and reconstruction formula that allowN-dimensional labeling distributions to be obtained from two-dimensional fluorescence distributions. The general problem ofM≥2 fluorochromes andN>M antibodies was shown to be reducible to the case of two fluorochromes. The method was tested by a triple labeling analysis of murine thymocytes.     

DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

Nonparametric flow cytometry analysis.

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.     

High-throughput microfluidic imaging flow cytometry

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Phytoplankton monitoring by flow cytometry

The application of flow cytometry to the monitoring of phytoplanktonis demonstrated. A comparison is made with conventional approachesto phytoplankton monitoring: light microscopy for the determinationof species abundance, and chlorophyll a determination and insitu chlorophyll a measurement by fluorescence for the determinationof the biomass. Flow cytometric measurements correlate wellwith these conventional types of measurements, as has been shownby comparing a full year of monitoring data obtained at a fixedmonitoring location 10 km off the Dutch coast. Flow cytometrybridges the gap between labour-intensive, but highly informative,microscopic observations and simple biomass measurements withless information content: via flow cytometry optical data areobtained at high speed for individual particles, which can betranslated into biomass information. On the basis of the flowcytometric measurements, rough discrimination of phytoplanktonspecies groups is possible, particularly for the abundant species.Of crucial importance is careful calibration of the flow cytometer,to ensure quantitative and comparable measurements over a longperiod of time. Calibration and quality assurance aspects arecovered in detail. ³Present address: Akzo Nobel Central Research Laboratories Arnhem,Department CRL, PO Box 9300, NL-6800 SB Arnhem, The Netherlands