A Monolithic Dual-Color Total-Internal-Reflection-Based Chip for Highly Sensitive and High-Resolution Dual-Fluorescence Imaging

We report a dual-color total-internal-reflection (TIR)-based chip that can generate two overlapping evanescent fields with different wavelengths for simultaneous imaging of two types of fluorophores. We derived a general relationship among the dimensions of the components of the chip to guarantee the overlap of two evanescent fields. Optical simulation results also confirm the generation and overlap of two evanescent fields. Using Si bulk micromachining and poly(dimethylsiloxane) (PDMS) casting, our fabrication method integrates all miniaturized optical components into one monolithic PDMS chip. Thus, assembly is unnecessary, and misalignment is avoided. Our PDMS chip can be employed with various sample delivery platforms, such as glass slide, flow cell, microchannel, etc. We first demonstrated the capability of the chip by imaging TIR fluorescent spots of a mixture of two fluorophores, namely, fluorescein and tetramethylrhodamine. We then employed the chip to observe the Brownian motion of a mixture of nile-red and dragon-green 500-nm microbeads. Our chip could potentially be integrated into a micro-total analysis system for highly sensitive and high-resolution dual-fluorescence imaging applications.