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

Abstract

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...

Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices

“…The dispersed signal is projected onto a Hamamatsu 7260-01 32-channel multianode PMT linear array photodetector (Hamamatsu). The detailed specifications of the linear-array PMTemployed were provided in the Supporting Information of a recent report published in Cytometry Part A (12). …”

“…This type of optical arrangement is characteristic of an emerging class of spectral FC systems, which attempt to measure an approximation of the full spectrum emitted by every analyzed bioparticle. The measurements produced by a spectral system may represent fluorescence, Raman, or surface-enhanced Raman scattering characteristics (9–12). …”

Microfluidic Approaches to Fluorescence In Situ Hybridization (FISH) for Detecting RNA Targets in Single Cells

“…Most examples of chip-based flow cytometry detectors that we are aware of are limited to one or two lasers, and 2-3 fluorescence and scatter channels. An alternative approach that enables more spectral channels is to replace the cascade of filters and photomultiplier tubes with a spectrograph (e.g., a diffraction grating and a multianode PMT or CCD detector), enabling collection of an entire fluorescence spectrum for each cell, with multivariate curve analysis to resolve individual spectral components [8,20]. A variety of cell sorting modalities based on optical trapping [24], valving [5], deflection with a piezoelectric transducer [3], or electrokinetics [34] have been coupled to chip-based flow cytometry, and these are expected to be compatible with FISH detection.…”

Multiparametric Analysis of Screening Data: Growing Beyond the Single Dimension to Infinity and Beyond

“…20 Further developments of the detector arrays of FACS machines have allowed the development of machines capable to monitoring up to 44 variables simultaneously on each particle detected in the flow cytometer. 21 Antibodies labeled with rare earth elements have allowed the development of mass cytometry [or time-of-flight mass cytometry (CyTOF)], in which up to 100 parameters can be detected on a cell-by-cell basis. In addition, by using specific reagents to label the protein content of the cell, this method allows multiplexing of samples for greater throughput.…”