TY - CONF A1 - Kage, Daniel A1 - Hoffmann, Katrin A1 - Wittkamp, M. A1 - Ameskamp, J. A1 - Göhde, W. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Dye-stained lifetime-encoded polymer microbeads for application in time-resolved flow cytometry N2 - Flow cytometry is a standard analytical tool for biological research and in medical applications. There are different requirements triggering recent device and method development depending on the desired field of application. One trend is governed by the need for an increasing number of simultaneously detectable codes, i.e., fluorescent labels. The other one focuses on cost-effective methods and development of miniaturized, portable devices. Fluorophore encoding is usually based on spectral encoding. However, this approach is hampered by, e.g., spectral crosstalk. Additionally, the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration limits the achievable number of detection channels. Moreover, spectral multiplexing typically requires several costly excitation light sources. Lifetime multiplexing and the discrimination between different encoding fluorophores and carrier beads based on their fluorescence decay kinetics could present an innovative alternative. Encoded beads, i.e., beads with lifetime codes corresponding to the surface chemistry, have been employed to evaluate the feasibility of this approach with a custom designed flow cytometer equipped with a pulsed light source and a fast detector for time-resolved measurements in a flow. In a first step, we used steady state and time-resolved photoluminescence measurements for the spectroscopic characterization of micrometer-sized dye-stained PMMA beads. Subsequently, the potential use of these microbeads for flow cytometry applications was analyzed with a prototype flow cytometer with lifetime detection. With our proof-of-concept studies, we could demonstrate that lifetime discrimination and simultaneous readout of a ligand fluorescence signal for analyte quantification is feasible with a set of dye-stained polymer microbeads at single wavelength excitation. These studies are expected to pave the road for new applications of fluorescence lifetime multiplexing in time-domain flow cytometry and bead-based assays in general. T2 - DBS/EBS 2017 CY - Potsdam, Germany DA - 20.03.2017 KW - Flow cytometry KW - Fluorescence KW - Life sciences KW - Lifetime encoding KW - Polymer particles PY - 2017 AN - OPUS4-39996 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kage, Daniel A1 - Hoffmann, Katrin A1 - Resch-Genger, Ute T1 - Luminophores for time-resolved flow cytometry N2 - 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. T2 - FLiMFlow meeting CY - Münster, Germany DA - 03.03.2017 KW - Flow cytometry KW - Fluorescence KW - Life sciences KW - Lifetime encoding KW - Polymer particles PY - 2017 AN - OPUS4-39307 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kage, Daniel A1 - Fischer, Linn A1 - Hoffmann, Katrin A1 - Wittkamp, M. A1 - Ameskamp, J. A1 - Göhde, W. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Stained microparticles for lifetime encoding - Flow cytometry application & photophysics N2 - 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. T2 - DPG Spring Meeting 2017 CY - Dresden, Germany DA - 19.03.2017 KW - Flow cytometry KW - Fluorescence KW - Life sciences KW - Lifetime encoding KW - Polymer particles PY - 2017 AN - OPUS4-40056 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kage, Daniel A1 - Hoffmann, Katrin A1 - Witkamp, M. A1 - Ameskamp, J. A1 - Göhde, W. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Time domain flow cytometry utilizing lifetime-encoded polymer microparticles N2 - 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. T2 - ANAKON 2017 CY - Tübingen, Germany DA - 03.04.2017 KW - Flow cytometry KW - Fluorescence KW - Life sciences KW - Lifetime encoding KW - Polymer particles PY - 2017 AN - OPUS4-39995 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -