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

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