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SPAD-Based Fluorescence Lifetime Analysis in Flow Cytometry
Summary
Scientists built a compact, affordable device (under $3,000) that can rapidly analyze cells by measuring how long they glow after being hit with a laser—a technique that gives extra detail beyond standard cell-sorting tools but has been too slow and expensive for widespread use. This new tool can screen up to 150,000 cells per second and can even detect microplastics without needing special dyes, which could help researchers better understand how these tiny plastic particles interact with human cells and tissues.
Flow cytometry (FCM) is an essential tool in medicine and biology for rapidly analyzing large cell populations. While cells are typically analyzed by their emission spectrum (ES), the analysis of the fluorescence lifetime (LT) provides valuable complementary information. However, the LT is rarely used because it requires efficient detection with high temporal resolution and ultrashort-pulsed excitation. The technical progress of Single-Photon Avalanche Diodes (SPADs) provides the potential to overcome these technical barriers and in this way make LT analysis increasingly feasible for high-throughput applications. To exploit this potential, the dependencies and limitations of SPAD-based fluorescence lifetime flow cytometry (LT-FCM) are investigated in this work through analytical and experimental analysis. This provides the background for nanosecond laser excitation and defines design guidelines for the setup and the detectors as well as the inherent trade-off between throughput and accuracy by a minimum of 100 detected photons and a maximum count rate of 0.46 photons per pixel and measurement window. In this way, SPADs enable high-throughput LT-FCM up to ~150,000 cells/s and compact, cost-efficient, and robust setups, demonstrated by the developed system in shoebox size (26 x 31 x 12 cm) and below 3000 €. Furthermore, the limited dynamic range of SPADs is identified as a major constraint for LT-FCM due to the varying intensity of cells. To overcome this limitation, a novel delay-control method is introduced that substantially extends the dynamic range and enables accurate LT determination even at 99 % detector saturation. These results significantly enhance the application potential of SPAD-based LT-FCM, demonstrated by standard cell analysis and label-free microplastic detection using the developed setup. Beyond LT-FCM, the SPAD pixel architecture enables combined LT and ES measurements in FCM. This approach requires advanced analysis methods, which are developed in this work and allow for rapid and precise determination of fluorophore fractions with a mean absolute error as low as 1.4 % and plastic materials with a differentiation accuracy of up to 95.7 %. In this way, this work provides a comprehensive framework for the widespread application of LT-FCM and its promising extension through combined spectral and temporal fluorescence analysis.