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Stanford University

Stanford Microfluidics Laboratory

Bleached-Fluorescence Imaging for Electrokinetics

Detailed flow visualizations help characterize system performance, identify non-ideal flow characteristics, and validate simulation efforts. In electrokinetic systems, quantities of interest include the electrophoretic mobility of samples, the diffusivity of samples, and the velocity of the bulk liquid. We have developed a flow visualization technique which we term photobleached-fluorescence imaging for visualizing microscale flow fields.

Project Description
The photobleached-fluorescence imaging technique can be used to obtain both molecular diffusion and advection information. The technique tracks fluorophores in the region of a photobleached line in a planar microdevice and yields quantitative diffusive and advective transport data. Visualizations of two- and weakly three-dimensional pressure- and electroosmotically-driven fluid flow fields have been demonstrated using the photobleaching of fluorescein and fluorescein-dextran conjugates. Photobleached-fluorescence imaging tracks undisturbed fluorophores, functions in polymer and glass microfluidic devices, can take advantage of fluorescent conjugates present in biochemical assays, and has a photobleached region that is flow independent. Below are example applications of the technique to quantify diffusivity, electrophoretic mobility, and pressure-driven flow effects, respectively.

Figure 1. Plot shows the evolution of a photobleached line in steady electroosmotic flow field in a 50 50 mm square cross-section capillary. The electrolyte used here is a dilute solution of fluorescein (< 1 mM) in 20 mM borate buffer. The bleached band moves through the capillary at 220 mm/s, and images have been centered about the band (applied electric field is 80 V/cm). Images were acquired with a high speed CCD camera with a frame rate of 53 frames per second. This time sequence is created from vertically-averaged axial intensity profiles for this simple straight-channel electroosmotic flow experiment. These profile intensities are of the form (Io-I), where I is the intensity of the imaged, unbleached fluorophores and Io is a normalization constant.

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Figure 2. Movie of the electrokinetic (including both electroosmotic and electrophoretic components) advection of a photobleached line in a 50 x 50 mm cross-section capillary (wall thickness = 25 mm) immediately after photobleaching. The fluid was a 20 mM borate buffer (pH 9) with a 1 mM concentration of fluorescein. The camera exposure time was 16.4 ms and the photobleaching pulse duration was 10 ms. The electric field was 80 V/cm. Pressure-driven flow was minimized by balancing the heights of reservoir fluid.

(Mouseover figure to begin movie)

Figure 3. Movie of combined bleached line subjected to a pressure-driven flow (from right to left) in the microchannel systems described above.


Mosier, B.M., Molho, J.I., Santiago, J.G., and Mikkelsen, J.C., "Bleached-Fluorescence Imaging of Microflows," in press, Experiments in Fluids, 2002.