Three-dimensional molecular mapping in a microfluidic mixing device using fluorescence lifetime imaging

Fluorescence lifetime imaging (FLIM) is used to quantitatively map the concentration of a small molecule in three dimensions in a microfluidic mixing device. The resulting experimental data are compared with computational fluid-dynamics (CFD) simulations. A line-scanning semiconfocal FLIM microscope allows the full mixing profile to be imaged in a single scan with submicrometer resolution over an arbitrary channel length from the point of confluence. Following experimental and CFD optimization, mixing times down to 1.3±0.4 ms were achieved with the single-layer microfluidic device. © 2008 Optical Society of America OCIS codes: 170.6920, 180.2520. Microfluidic systems are attractive for studying reaction kinetics in fluids because of the potential to reduce reagent volumes, time and cost of fabrication, and mixing speeds [1]. To allow accurate measure ment of rate constants using a microfluidic mixer, it is important to obtain a comprehensive understanding of the fluid dynamics within the system. It is standard practice to simulate a mixing device design using computational fluid dynamics (CFD) to optimize the microfluidic device and also assist in the interpretation of kinetic data [2]. However, it is normally extremely challenging to validate the CFD simulations. Herein we present a method for directly visualizing fluid dynamics in a microfluidic mixer using optically sectioned fluorescence-lifetime imaging (FLIM) and compare these experimentally obtained results with three-dimensional (3-D) CFD simulations. Previously, steady-state fluorescence has been employed to monitor the mixing of fluids within hydrodynamic focusing devices. These reports have included the use of Forster resonance energy transfer [3], two-photon absorption [4,5], and confocal fluorescence imaging [6]. Although such methods have proven to be successful, intensity imaging is prone to artefacts associated with fluorophore concentration, nonuniform illumination, detection efficiencies, the inner-filter effect, and optical scattering. FLIM has already been shown to provide a robust means of imaging mixing in microfluidic environments [4,7]. Here we demonstrate a novel scheme to provide 3-D chemical concentration mapping over an extended cm field of view. This involves a single automated data-acquisition step that images the (calibrated) change in lifetime due to the presence of a molecular quenching agent [8]. We compare the experimental data to the CFD simulations to validate our technique.