Single particle high resolution spectral analysis flow cytometry

Goddard, Gregory; Martin, John; Naivar, Mark A.; Goodwin, Peter M.; Graves, Steven W.; Habbersett, Robert C.; Nolan, John P.; Jett, James H.

doi:10.1002/cyto.a.20320

Cited by 54 publications

(61 citation statements)

References 45 publications

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“…More recently, in 2006, Goddard et al presented a system that uses a diffraction grating to disperse the collected fluorescence and side-scattered light and a CCD image sensor coupled to a spectrograph to collect the signals (15). The system was built around the flow cell and collection optics of a conventional flow cytometer with minimal modifications.…”

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...

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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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“…Additionally, this assay measures total DNA but cannot be able to differentiate between intact human DNA and bacterial or fungal DNA. Several modifications to flow cytometry have been made due to which it become more specific and sensitive (Goddard et al 2006), but the approaches have not extended the general acceptance. Therefore, this technique is not commonly used for forensic purposes.…”

Section: Introductionmentioning

confidence: 99%

An introduction to the single cell gel electrophoresis assay: a technique resolving issues in forensic science

Shukla

2018

Egypt J Forensic Sci

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Time since death (TSD) estimation is a crucial issue in death investigation. In majority of homicide/suicide cases deceased body recovered within the first 48 h, therefore, it is critically important to determine the time of death quickly and precisely. In the present scenario, TSD estimation still remains difficult even for experienced pathologists undertaken with extreme caution. After death numerous factors may be of cadaveric or environmental origin can influence the 'normal' rate of postmortem changes. Therefore, it could be more difficult to estimate longer TSD. However, these environmental influencing factors can also effect on DNA in the form of degradation, which start after 1-2 h of death. This DNA degradation could be work as molecular clock which help to estimate early TSD. Single-cell gel electrophoresis (SCGE), also known as the Comet assay, is the technique by which DNA degradation can be visualized and measured qualitatively as well as quantitatively. Using this assay not only early TSD estimated but many other challenges such as time since deposition of biological fluids, repair of genetic material from degraded biological sample could also be resolved. With the help of this paper an attempt was made to introduce a well-known cytogenetic technique that is SCGE assay, which could be a versatile technique for forensic science.

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“…For example, Goddard et al [26] utilized a diffraction grating to disperse the fluorescence signals emitted from the cells or microspheres passing through the interrogation region of the cytometer, and then used a CCD image sensor to analyze the spectral distribution of the collected signals. Individual fluorophores in multiple-tagged samples were successfully detected with high resolution.…”

Section: Introductionmentioning

confidence: 99%

Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection

Lin

2016

Applied Sciences

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A high performance diascopic illumination configuration is presented for the simultaneous detection of cells and particles with different sizes and different fluorescence labels in a microchannel. In the proposed approach, the cells/particles are illuminated by an objective-type dark-field condenser equipped with a low-cost tungsten light source and are then characterized by extracting the side-scatter, absorbance, and fluorescence signals from the spectra obtained by a ultraviolet-visible-near infrared (UV-Vis-NIR) spectrometer. A modified computation model is adopted to improve the capability for discriminating more fluorescence dyes simultaneously. The feasibility of the proposed detection configuration is demonstrated by counting and classifying a mixed sample of green, red, and crimson fluorescent-labeled particles and non-labeled particles with various dimensions. The suitability of the proposed system for real-world cytometry applications is then evaluated by classifying a mixed bio-sample comprising of gastric epithelial (AGS) cells stained with Trypan-blue and Erythrosin-bluish dye, respectively. The results show that the cytometer enables the efficient detection, identification, and classification of mixed bio-samples without the need for spatial filters or delicate optical components. Consequently, the proposed system has significant potential for high-performance micro-flow cytometry applications.

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Single particle high resolution spectral analysis flow cytometry

Cited by 54 publications

References 45 publications

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

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

An introduction to the single cell gel electrophoresis assay: a technique resolving issues in forensic science

Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection

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