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Hyperspectral cytometry at the single‐cell level using a 32‐channel photodetector
Author(s) -
Grégori Gérald,
Patsekin Valery,
Rajwa Bartek,
Jones James,
Ragheb Kathy,
Holdman Cheryl,
Robinson J. Paul
Publication year - 2012
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.21120
Subject(s) - hyperspectral imaging , cytometry , detector , single cell analysis , fluorescence , multiplexing , flow cytometry , computer science , optics , materials science , biological system , physics , optoelectronics , chemistry , artificial intelligence , telecommunications , biology , cell , biochemistry , genetics
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. © 2011 International Society for Advancement of Cytometry