A 40-MHZ FREQUENCY MULTIPLEXED ELECTRONIC SYSTEM FOR MULTICOLOR DROPLET FLOW CYTOMETRY Khaled M. Dadesh 1,* and Amar S. Basu 1, 2, ** 1 Electrical and Computer Engineering Department, 2 Biomedical Engineering Department, Wayne State University, Detroit MI ABSTRACT Multiparameter flow cytometry is commonly used for the analysis of cells and droplet microreactors. However, their cost and complexity limits its applications in point-of-care systems. This paper demonstrates a high speed frequency multiplexed electronic detector for multicolor flow cytometry. The system can detect multiple fluorophores on a single PMT, rather than one per fluorophore. The detector is operating at up to 40MHz, enabling a multi-channel bandwidth of >200 KHz as required by cell cytometry. The system successfully distinguishes alternating water-in-oil droplets containing fluorescein or Alex 680 alternately at throughputs of 300 drops/sec and single fluorescein droplets at 2800 drop/sec. KEYWORDS: LIF, Multiplexed detection, fluorimetry, fluorescence, lock in amplification, droplets INTRODUCTION Flow cytometry is capable of analyzing >200,000 cells/sec and is used in many applications, including the diagnosis of HIV and leukemia [1-4]. It has also been used for the high throughput analysis of droplet microreactors [5]. Multicolor flow cytometry is useful for distinguishing heterogeneous cell or droplet populations; however, they are considerably more expensive because they require multiple photomultiplier tubes (PMTs), high voltage supplies, and optical paths. Previously, we demonstrated a novel LIF detection system which uses frequency division multiplexing (FDM) for detecting multiple fluorophores. This system requires only a single PMT and optical path, resulting in significant cost savings. However, the modulation frequency of the system (1.5 KHz) resulted in a measurement speed of only ~1-10 drops/sec [6]. Here, we report a new circuit topology which improves the measurement bandwidth by >30,000X, enabling multicolor detection at 200 KHz as needed for high speed flow cytometry of cells and droplets. SYSTEM CONCEPT AND OPERATION Fig. 1 shows the flow cytometry circuit and the system integrated in an inverted microscope. Three independent modulation circuits (Fig. 1A) modulate three excitation laser diodes (450/520/680nm) at unique carrier frequencies: 25MHz, 32MHz, and 40MHz, respectively. The lasers are combined using the device shown in Fig. 2, directed into the fluorescence port of an inverted epi-fluorescence microscope, and focused into the sample by a 40X objective. Consequently, each fluorophore in the detection window is excited at a unique modulation frequency. Figure 1: A) Printed circuit board of the final FDM system B) Integration of the FDM circuit and optics in a conventional inverted microscope. This shows an example of a two color FDM system with red and blue channels. The fluorescence emissions are directed through a multi-band-pass emission filter (Fig. 3) to remove the excitation signals, and are then detected by a single PMT. The PMT signal, containing all three fluorescence channels, is then de- multiplexed using a heterodyne demodulator circuit. This heterodyne demodulator emulates a multi-channel lock-in detector. B A 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 910 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany