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For the application of time-resolved detection in the framework of flow cytometry, suitable luminescence lifetime code carriers are required. Here we report on our achievements concerning strategies to increase the accessible range of lifetime values and to realize continuously tunable lifetimes.
To that end, we investigated polymer (PMMA) microbeads stained with mixtures of dyes exhibiting different fluorescence decay kinetics. At the expense of spectrally varying decay kinetics, it is possible to modify the lifetime by changing the dye concentration ratio.
Moreover, semiconductor quantum dots incorporated into polymer beads were studied as alternative luminophores outperforming organic dyes with respect to long luminescence lifetimes, flexible choice of excitation wavelength and narrow spectral emission width.
Our experiments demonstrate that lifetime adaption with dye mixing is basically feasible and semiconductor quantum dots represent promising candidates for long-lifetime codes.
Multiparametric analyses involving optical techniques like flow cytometry are at the core of studying complex systems in biological research and diagnostic applications. However, for fluorescence-based techniques, the number of reporters distinguishable in spectral multiplexing is limited by spectral overlap and requires a multitude of excitation light sources and detection Systems. Intensity encoding often used for bead assays suffers from problems regarding dye concentration control and excitation light intensity fluctuations. An alternative is luminescence lifetime encoding, particularly to minimize instrument costs.
Here, we report on dye-stained polymer microparticles for lifetime encoding in flow cytometry with different organic dyes. This ranges from studies of the impact of parameters like dye loading concentration and particle diameter on fluorescence decay behavior to the demonstration of lifetime code reading and simultaneous ligand fluorescence signal detection with single-wavelength excitation in a flow.
Tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads
(2020)
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 is a widely used method in biological research and medical diagnostics. Depending on the respective application, two opposing directions of development are currently of interest. On the one hand, there is a need for analyses of growing complexity employing more and more fluorescent labels and codes. On the other hand, cost-effective methods and portable, miniaturized, and robust instruments are desired.
Commonly performed spectral multiplexing utilizing a color code suffers from several problems, such as the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration and hence, photobleaching, dye leaking for certain encoding procedures, and spectral crosstalk, limiting the achievable number of detection channels. Moreover, it typically requires several costly excitation light sources. An innovative alternative can be lifetime multiplexing and the discrimination between different encoding fluorophores and carrier beads based on their fluorescence decay kinetics. In order to examine the potential of this approach, dye encoded beads (lifetime encoded surface chemistry) were prepared using several fluorophores from different dye classes and their suitability for lifetime discrimination in a flow was tested in conjunction with a custom designed flow cytometer equipped with a pulsed light source and a fast detector.
In a first step, the spectroscopic properties of micrometer-sized dye-stained PMMA beads were studied by means of steady state and time-resolved photoluminescence measurements. For the performance of studies on the practical use of these microbeads in flow cytometry applications, a custom-built demonstrator model for a flow system was employed.
Our results demonstrated that lifetime discrimination and simultaneous readout of a ligand fluorescence signal for analyte quantification with a set of dye-stained polymer microbeads at single wavelength excitation is feasible. These studies are expected to pave the road for new applications of fluorescence lifetime multiplexing within the framework of time-domain flow cytometry and bead-based assays.