A perfusable microfluidic device with on-chip total internal reflection fluorescence microscopy (TIRFM) for in situ and real-time monitoring of live cells
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A microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis was developed. This integrated device enables in situ Total Internal Reflection Fluorescence Microscopy (TIRFM) on adherent cells cultured under continuous medium perfusion. This TIR-based chip, allows TIRFM to be easily performed on cells without the assembly of complicated optical components and cell culture chambers. The integrated device was evaluated by tracking the movement of fluorescent beads and monitoring the location of insulin granules in mouse pancreatic β-cells. This system offers higher signal-to-noise (S/N) ratio than epi-fluorescence microscopy (EPIFM), and comparable image quality to commercial TIRFM systems when imaging insulin granules. We also detected repetitive changes in intracellular Ca2+ concentration in MIN6-m9 cells stimulated with KCl, which demonstrates quick perfusion for cell analysis while maintaining high S/N ratio.
KeywordsTotal internal reflection fluorescent microscopy (TIRFM) Cell analysis Microfluidics
This research was partially supported by Core Research for Evolutional Science & Technology (CREST), Japan Science and Technology Agency (JST), Japan. We thank Dr. N. C. H. Le with CSIRO Materials Science and Engineering, Australia for useful advice, Prof. S. Seino with Kobe University for providing MIN6-m9 cells, Prof. S. Nagamatsu with Kyorin University, Japan for providing GFP-labeled insulin vector, and Prof. H. Yokota with Kyoto University for the use of Nikon TIRFM system.
- K. Minami, H. Yano, T. Miki, K. Nagashima, C.Z. Wang, H. Tanaka, J.I. Miyazaki, S. Seino, Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines. Am. J. Physiol. Endocrinol. Metab. 279(4), E773–E781 (2000)Google Scholar
- M. Ohara-Imaizumi, C. Nishiwaki, T. Kikuta, S. Nagai, Y. Nakamichi, S. Nagamatsu, TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic ƒÀ-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat ƒÀ-cells. Biochem. J. 381(Pt 1), 13 (2004)Google Scholar
- C.M. Zettner, M. Yoda, Particle velocity field measurements in a near-wall flow using evanescent wave illumination. Exp. Fluid. 34(1), 115–121 (2003)Google Scholar