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.

Aggregation-induced emission (AIE) has been meanwhile observed for many dye classes and particularly for fluorophores containing propeller-like groups. Herein, we report on the AIE characteristics of a series of four hydrophobic pyrrolidinylvinylquinoxaline (PVQ) derivatives with phenyl, pyrrolyl, indolyl, and methoxythienyl substituents used to systematically vary the torsion angle between this substituent at the quinoxaline C2 position and the planar PVQ moiety. These molecules, which are accessible via four- or five-component one-pot syntheses, were spectroscopically studied in organic solvents and solvent–water mixtures, as dye aggregates, solids, and entrapped in polystyrene particles (PSP). Steady-state and time-resolved fluorescence measurements revealed a strong fluorescence enhancement for all dyes in ethanol–water mixtures of high water content, accompanying the formation of dye aggregates with sizes of a few hundred nm, overcoming polarity and H-bonding-induced fluorescence quenching of the charge-transfer-type emission of these PVQ dyes. The size and shape of these dye aggregates and the size of the AIE effect are controlled by the water content and the substituent-dependent torsion angle that influences the nucleation process and the packing of the molecules during aggregation. Staining of 1 μm-sized carboxy-functionalized PSP with the PVQ dyes resulted also in a considerable increase in the fluorescence quantum yield and lifetime, reflecting the combined influence of the restricted molecular motion and the reduced polarity of the dye microenvironment.

Semiconductor quantum dots (QDs) embedded into polymer microbeads are known to be very attractive emitters for spectral multiplexing and colour encoding. Their luminescence lifetimes or decay kinetics have been, however, rarely exploited as encoding parameter, although they cover time ranges which are not easily accessible with other luminophores. We demonstrate here the potential of QDs made from II/VI semiconductors with luminescence lifetimes of several 10 ns to expand the lifetime range of organic encoding luminophores in multiplexing applications using time-resolved flow cytometry (LT-FCM). For this purpose, two different types of QD-loaded beads were prepared and characterized by photoluminescence measurements on the ensemble level and by single-particle confocal laser scanning microscopy. Subsequently, these lifetime-encoded microbeads were combined with dye-encoded microparticles in systematic studies to demonstrate the potential of these QDs to increase the number of lifetime codes for lifetime multiplexing and combined multiplexing in the time and colour domain (tempo-spectral multiplexing). These studies were done with a recently developed novel luminescence lifetime flow cytometer (LT-FCM setup) operating in the time-domain, that presents an alternative to reports on phase-sensitive lifetime detection in flow cytometry.Flow cytometry (FCM), where either single cells or particles are optically detected in a flow, is an established and widespread multiparametric fluorescence technique used for many routine and research applications in biology, medical diagnostics, and food analysis 1-5 . FCM typically relies on organic luminophores for optical encoding and multiplexing as well as analyte quantification 6-10 . State-of-the-art FCM instruments are designed to detect spectral and/or intensity codes and can distinguish a large number of different colour and intensity codes, e.g. in the case of bead-based assays 5,11-13 . 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] . The concept of lifetime multiplexing in a flow, however, has not been adopted in routine applications up to now due to the limited measurement time per object resulting in reduced photon count numbers 27,29 . Moreover, the vast majority of reports on lifetime detection in flow cytometry relies on frequency-domain techniques (phase fluorometry), which used to be less expensive as f...