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Anal Chem. 2019 Feb 19;91(4):3055-3061. doi: 10.1021/acs.analchem.8b05451. Epub 2019 Feb 8.

One Sensor for Multiple Colors: Fluorescence Analysis of Microdroplets in Microbiological Screenings by Frequency-Division Multiplexing.

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Bio Pilot Plant , Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute , 07745 Jena , Germany.
Faculty of Biology and Pharmacy , Friedrich Schiller University , 07743 Jena , Germany.
Ilmenau University of Technology , 98693 Ilmenau , Germany.
LaCoSys GmbH , 07745 Jena , Germany.


High-speed multiwavelength fluorescence measurements are of paramount importance in microfluidic analytics. However, multicolor detection requires an intricate arrangement of multiple detectors and meticulously aligned filters and dichroic beamsplitters that counteract the simplicity, versatility, and low cost of microfluidic approaches. To break free from the restrictions of optical setup complexity, we introduce a simpler single-sensor setup based on laser-frequency modulation and frequency-division multiplexing (FDM). We modulate lasers to excite the sample with four non-overlapping frequency signals. A single photomultiplier tube detects all the modulated emitted light collected by an optical fiber in the microfluidic chip. Signal demodulation is performed with a lock-in amplifier separating the emitted light into four color channels in real time. This approach not only reduces complexity and provides setup flexibility but also results in improved signal quality and, thus, higher signal-to-noise ratios that translate into increased sensitivity. To validate the setup for high-throughput biological applications, we measured multiple signals from different microorganisms and fluorescently encoded droplet populations for exploring beneficial or antagonistic roles in microbial cocultivation systems, as is the case for antibiotic screening assays.

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