Highly-sensitive fluorescence detection and imaging with microfabricated total internal reflection (TIR)-based devices


This paper presents the development and application of several total internal reflection (TIR)-based devices for highly-sensitive and high-resolution fluorescence imaging. Using micro electro mechanical systems (MEMS) fabrication technology, miniaturized single-wavelength and dual-wavelength TIR-based devices have been designed and fabricated. A low-cost and simple fabrication process utilizing TMAH wet etching, deep reactive ion etching (DRIE) and polymer casting has enabled us to integrate several optical components into one single poly(dimethylsiloxane) (PDMS) chip, thus alignment and assembly are eliminated. The slide-format and monolithic chip can be used with both upright and inverted fluorescent microscope with interchangeable sample delivery platforms, i.e. glass slides, flow-cells, microchannels etc…In different configurations, the devices were applied in measurement of the average velocity field of fluorescent beads in pressure-driven flow in a microchannel and then in detecting of single DNA molecules. Another ongoing effort is focused on using the device for cell imaging which will be briefly discussed. These applications are the first step toward integration the TIR-based chips into a detection functionality of a μ-TAS. Our proposed devices are smaller, simpler and less expensive comparing to current objective-type and prism-type total internal reflection fluorescent microscopy (TIRFM) systems. At this moment, our devices could provide quick and low-cost evanescent excitations with one or two wavelengths in upright or inverted fluorescent microscopes using normal objective lenses. In the future, they might be integrated as a detection module in a μ-TAS where highly-sensitivity and high-resolution fluorescence imaging capability is necessary. Finally, some discussion on the limitation and future developments for our devices will be also provided.

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N. C. H. Le gratefully acknowledges the Ministry of Education, Culture, Sports, Science and Technology, Government of Japan (MEXT) of Japan for his doctoral scholarship. This study was partially supported by the Kyoto NanoTech Cluster and Research for Promoting Technological Seeds grant from Japan Science and Technology Agency (JST). The authors thank Prof. M. Kasahara with Department of Bioscience and Biotechnology, Ritsumeikan University for helping the preparation of DNA fragments.

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Correspondence to Nam Cao Hoai Le.

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Le, N.C.H., Dao, D.V., Yokokawa, R. et al. Highly-sensitive fluorescence detection and imaging with microfabricated total internal reflection (TIR)-based devices. J. Micro-Nano Mech. 7, 45–59 (2012). https://doi.org/10.1007/s12213-012-0043-3

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  • Total internal reflection (TIR)-based chip
  • Fluorescent imaging
  • Micro electro mechanical systems (MEMS)
  • Single molecule detection
  • Nano-particle image velocimetry (nano-PIV)
  • Cell imaging
  • Micro-total analysis systems (micro-TAS)