&lt;title&gt;Real-time Fourier transform spectrometry for fluorescence imaging and flow cytometry&lt;/title&gt;

Buican, Tudor N.

doi:10.1117/12.17787

Cited by 22 publications

(14 citation statements)

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“…Approach Comments Wade et al (1979) Grating spectrograph and silicon-intensified detector Measured average spectra of many cells Steen and Stokke (1986) Grating monochromometer and PMT Measured average intensity at 10-nm intervals Buican (1990) Fourier transform FC Gauci et al (1996) Prism and intensified photodiode array Measured spectra of single cells and microspheres Fuller and Sweedler (1996) Grating spectrograph and CCD Measured spectra of single microspheres and liposomes Asbury et al (1996) Scanning monochromometer and PMT…”

Section: Referencementioning

confidence: 99%

Spectral Flow Cytometry

2013

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Interest in measuring the complete fluorescence spectra of individual cells in flow can be traced to the earliest days of flow cytometry. Recent advances in detectors, optics, and computation have made it possible to make full spectral measurements in the sub‐millisecond time frame in which flow cytometry measurements typically occur. This opens up new possibilities for applying spectroscopy to the analysis of individual cells. This unit reviews historical and contemporary approaches to spectral flow cytometry, as well as instrument design, calibration, and data analysis for spectral flow cytometry applications. Curr. Protoc. Cytom. 63:1.27.1‐1.27.13. © 2013 by John Wiley & Sons, Inc.

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Section: Referencementioning

confidence: 99%

Spectral Flow Cytometry

2013

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“…Most spectral imaging of two-dimensional scenes is presently performed with tunable filters [1][2][3][4] , interferometers 5,6 , or dispersive apparatus 7 to measure or calculate an image cube comprising a spatial representation of the scene at a variety of wavelengths. Subsequent computer analysis such as classification or pixel unmixing is used to determine the information of interest.…”

Section: Introductionmentioning

confidence: 99%

<title>Beyond image cubes: an agile lamp for practical 100% photon-efficient spectral imaging</title>

Miller¹,

Levenson²

2001

Biomarkers and Biological Spectral Imaging

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Hardware for obtaining spectral image cubes using filters or interferometers, though capable of revealing subtle aspects of composition, is typically expensive, bulky, slow, and often provides poor spatial resolution. In addition, a great deal of computer processing is needed to extract information from the raw data: interferometers must perform FFTs on megapixel data sets; all approaches involve calculations of spectral indices in order to classify or analyze the scene into its components. Consequently, spectral imaging techniques have been adopted only by a small, pioneering community.We report on a novel agile lamp for imaging which produces illumination having any desired spectral flux distribution ranging from pure spectral bands to precisely-tailored, complex polychromatic functions. This lamp, together with a CCD camera, is suitable for use in most spectral imaging applications, and by enabling one to directly image the scene in the spectral measure of interest, it eliminates the need for computer processing. Using matched filtering, one can obtain full information from all spectral bands in a handful of exposures with optimal signal-to-noise. The lamp is long-lived and spectrally stable. It will be affordable, compact, and rugged, and its spectral output can be adjusted within one millisecond. Finally, as there is no interferometer or other optics involved, imaging is 100% photon-efficient. Use of spectrally agile lamps in various multispectral applications is expected.

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“…Steen and Stokke measured averaged fluorescence spectra of rat thymocytes in 1986, using a custom-built cytometer and grating monochromator (9,10). In 1990, Buican used a Fourier-transform interferometer to obtain singlecell spectra (11). The performance of his design was severely limited by the fact that cells needed to stay in the laser beam for a relatively long time to be scanned; modern high-speed flow cytometers use a laser excitation time of $1-10 ls.…”

Section: Flow Cytometry (Fcm) Is a Very Powerful Cell-analysis Technimentioning

confidence: 99%

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

et al. 2011

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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 wellseparated 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 ls. 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. ' 2011 International Society for Advancement of Cytometry Key terms hyperspectral cytometry; flow cytometry; next-generation instruments FLOW cytometry (FCM) is a very powerful cell-analysis technique, applied in various fields of life science ranging from basic cell biology to genetics, immunology, molecular biology, microbiology, plant cell biology, and environmental science (1). FCM uses optical properties of biological particles and makes analysis possible at the single-cell level. Forward-angle light scatter (size-related) and side-angle light scatter (shape-and structure-related) as well as various fluorescence emissions are collected following illumination/excitation (usually by one or several lasers). The data are collected, digitized, and stored on a computer where they are further processed to discriminate populations of particles (cells) with similar characteristics.Detection systems used in current commercial instruments are almost all based on a simple con...

show abstract

<title>Real-time Fourier transform spectrometry for fluorescence imaging and flow cytometry</title>

Cited by 22 publications

References 1 publication

Spectral Flow Cytometry

Spectral Flow Cytometry

<title>Beyond image cubes: an agile lamp for practical 100% photon-efficient spectral imaging</title>

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

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