A novel ratiometric method for determining the consequences of cell-size features in a concentration gradient generator
We present a multi-dye ratiometric method for the correction of optical artifacts and unequal illumination encountered by researchers that utilize complex microfluidic systems. Using a novel chemotaxis system that provides cells with passively generated chemoattractant gradients, we demonstrate that the currently utilized method of single-color epi-fluorescence is limited in its ability to characterize gradient formation in the presence of differing channel heights and in the proximity of micron-sized features. As future devices strive to mimic the microtopography of physiological environments, this deficit will become increasingly relevant. The presented multi-color methodology allows for the correction of standard wide-field images and, with the incorporation of laser scanning confocal microscopy, explores three-dimensionally resolved gradient analysis. Using a validated numerical model, we also analyze the potential distortion in gradient formation introduced by device microstructure and the very cells that the system is designed to examine. Our analysis has important implications for the sensitivity of microfabricated systems to cell loading density and channel aspect ratio. In conclusion, the imaging and modeling methodologies introduced in this work will provide complementary information for the rational design and validation of microfluidic devices with cell-sized features.