Time-domain single photon-excited autofluorescence lifetime for label-free detection of T cell activation.
Time-domain single photon-excited autofluorescence lifetime for label-free detection of T cell activation.
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DOI：
10.1364/ol.422445
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发表时间：
2021-05-01
期刊：
影响因子：
3.6
通讯作者：
Skala MC
中科院分区：
文献类型：
--
作者：
Samimi K;Contreras Guzman E;Trier SM;Pham DL;Qian T;Skala MC
Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique, capable of label-free assessment of metabolic state and function within single cells. FLIM measurements of autofluorescence were recently shown to be sensitive to functional state and subtype of T cells. Therefore, autofluorescence FLIM could improve cell manufacturing technologies for adoptive immunotherapy, which currently require a time-intensive process of cell labeling with fluorescent antibodies. However, current autofluorescence FLIM implementations are typically too slow, bulky, and prohibitively expensive for use in cell manufacturing pipelines. Here, we report a single photon-excited confocal whole-cell autofluorescence system that uses fast field-programmable gate array (FPGA)-based time tagging electronics to achieve time-correlated single photon counting (TCSPC) of single cell autofluorescence. The system includes simultaneous near-infrared bright-field imaging and is sensitive to variations in the fluorescence decay profile of the metabolic coenzyme NAD(P)H in human T cells due to activation state. Classification of activated and quiescent T cells achieved high accuracy and precision (ROC AUC = 0.92). The lower cost, higher acquisition speed, and resistance to pile up effects at high photon flux compared to traditional multiphoton-excited FLIM and TCSPC implementations with similar SNR makes this system attractive for integration into flow cytometry, sorting, and quality control in cell manufacturing.
