Lock-In Detection of Directivity of Fluorescence From a Minute Particle Using TAS Chip Incorporated With Radially Arranged Light Waveguides

Abstract

It has long been thought that an optical sensor, such as a light waveguide implemented total analysis system (TAS), is one of the most functional components that will be needed to realize a “ubiquitous human healthcare system”. A transparent resin-based TAS chip incorporated with a light waveguide [1] is quite preferable in such a cost-effective and disposal use. In line with the technical demand, we have already proposed a specially fabricated structure for an epoxy resin-based monolithic light waveguide capable of illuminating a cell or particle running along a microfluidic channel [2], as well as of obtaining directivity of fluorescence with a radially arranged waveguide structure (as shown in Figure 1) and a sequential light scanning mechanism based on a forced vibrated optical fiber [3]. Utilizing this TAS system, we have successfully detected preliminary results of fluorescence directivity of a 5-μm-diameter polystyrene particle with scanning angle range of 180 degrees, at illuminating light scanning frequency of approximately 1.7 kHz [4]. However, the transmittance of the trial-manufactured light waveguides was slightly lower owing mainly to its smaller cross section size, and, as a result, signal-to-noise ratio of detected fluorescence signal waveform was not as good as we have expected. To improve the S/N ratio, it is necessary to increase illuminating power of a laser source, and, at the same time, to increase multiplication factor of a photo-electron multiplier sensor to beyond its performance limit. Unfortunately, with the capability of the current equipment, it is difficult to drastically improve the S/N ratio. In this paper, we attempted to apply AC detection method to measure extremely weak fluorescence with a high frequency modulated laser source of its wavelength of 488 nm, and with a high speed lock-in-amplifier having both higher reference frequency up to 3MHz and smaller time constant.