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Flow cytometry visualization and real-Time processing with a CMOS SPAD array and high-speed hardware implementation algorithm

Research output: Chapter in Book/Report/Conference proceedingConference paper

Hanning Mai, Simon P. Poland, Francesco Mattioli Della Rocca, Conor Treacy, Justin Aluko, Jakub Nedbal, Ahmet T. Erdogan, Istvan Gyongy, Richard Walker, Simon M. Ameer-Beg, Robert K. Henderson

Original languageEnglish
Title of host publicationImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII
EditorsDaniel L. Farkas, Attila Tarnok
PublisherSPIE
ISBN (Electronic)9781510632493
DOIs
Publication statusPublished - 1 Jan 2020
EventImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII 2020 - San Francisco, United States
Duration: 3 Feb 20206 Feb 2020

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume11243
ISSN (Print)1605-7422

Conference

ConferenceImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII 2020
CountryUnited States
CitySan Francisco
Period3/02/20206/02/2020

King's Authors

Abstract

Time-domain microfluidic fluorescence lifetime flow cytometry enables observation of fluorescence decay of particles or cells over time using time-correlated single photon counting (TCSPC). This method requires the fluorescence lifetime measured from a limited number of photons and in a short amount of time. In current implementations of the technique, the low throughput of state of the art detectors and lack of real-Time statistical analysis of the current technology, the timedomain approaches are usually coupled with off-line analysis which impedes its use in flow cell sorting, tracking and capturing. In this work, we apply a 32×32 CMOS SPAD array (MegaFrame camera) for real-Time imaging flow cytometry analysis. This technology is integrated into a 1024-beam multifocal fluorescence microscope and incorporating a microfluidic chip at the sample plane enables imaging of cell flow and identification. Furthermore, the 1.5% native pixel fill-factor of the MegaFrame camera is overcome using beamlet reprojection with <10 μW laser power at 490 nm for each beam. Novel hardware algorithms incorporating the center-of-mass method (CMM) with real-Time background subtraction and division are implemented within the firmware, allowing lossless recording of TCSPC events at a 500 kHz frame rate with 1024 histogram bins at 52 ps time resolution. Live calculation of background compensated CMM-based fluorescence lifetime is realized at a user-defined frame rate (typically 0.001 ∼ 27 kHz) for each SPAD pixel. The work in this paper considers the application of the SPAD array to confocal fluorescence lifetime imaging of multiple coincident particles flowing within a microfluidic channel. Compared to previous flow systems based on single-point detectors, the multi-beam flow system enables visualization, detection and categorization of multiple groups of cells or particles according to their fluorescence lifetime.

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