Luminescence lifetime encoding in time-domain flow cytometry

Abstract

Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.

Tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads

“…2, the luminescence decays of both types of QD-encoded beads show a multi-exponential behaviour, with the mean lifetime increasing with increasing emission wavelength. Besides background contributions 21 , we tentatively attribute this observation to the presence of QDs with slightly different optical properties within the beads. It needs to be kept in mind that each QD ensemble contains nanocrystals of different size and most likely also slightly different surface chemistries, resulting in slightly different energy bands and emission maxima (see Fig.…”

“…resolution were performed with a recently developed Quantum P/pantau flow cytometer (Quantum Analysis GmbH, Germany). This LT-FCM setup was recently described in detail 21 . The instrument is equipped with a square-wave-modulated laser diode emitting at 488 nm (Nichia Corporation, Japan) with a repetition rate of 5 MHz (average output power was 50 mW), one detector for time-resolved photon counting with a time binning of 2.5 ns as well as three conventional PMTs in low-bandwidth mode for the side-scattered light channel and two fluorescence channels.…”

“…For lifetime determination in LT-FCM, the straightforward adaption of the integration time range θ in Eq. (1) can be used to drastically improve the discrimination capabilities 21 . The dependence of the mean lifetime mean τ of single-bead measurements on the time range θ measured with our LT-FCM setup is shown Fig.…”

“…The dependence of the lifetime on the time range can be explained by introducing a model function for the fluorescence decay in Eq. (1) 21 . In this case, we chose a general expression for multiexponential decays with amplitudes A j and constant background B and obtained Eq.…”

“…Commonly applied spectral multiplexing approaches utilizing organic luminophores are, however, limited by the spectral overlap of the emission bands of the different encoding dyes which limits the number of distinguishable codes and can require signal compensation and cross talk correction 6,14,15 . The exploitation of the luminophore-characteristic luminescence lifetime (LT) as additional encoding parameter could in principle increase the number of accessible codes in flow cytometry analysis, thereby increasing the degree of multiplexing 6,7,14,[16][17][18][19][20][21] . Time-resolved fluorescence measurements in flow cytometry and lifetime encoding have been discussed for decades 8,[22][23][24][25][26][27][28] .…”