A Live Cell Imaging Microfluidic Model for Studying Extravasation of Bloodborne Bacterial Pathogens

Figure 1

In vitro model to study endothelial transmigration of bacteria under physiological shear stress. (a) Microfluidic device, top view: PDMS input and collection channels sandwiching a porous, transparent membrane (blue box) coated with endothelia grown to 2 days postconfluence. White squares: imaging sites. (b) Cross-sectional schematic showing GFP-expressing B. burgdorferi (green) migrating from input to collection channels through endothelial monolayer stained with nontoxic live cell imaging plasma membrane dye (orange) and membrane (dashed black line). Red arrow: flow direction. At each imaging site, 3D -series encompassing the full depth of input (~200 μm) and collection (~600 μm) channels were acquired at ~7 fps (~26 fps in ) in 2 channels simultaneously, using a resonant scanning confocal microscope equipped with a high numerical aperture (NA) long working-distance (LWD) water-immersion objective. (c) Postconfluent endothelial monolayers in devices visualized by phase contrast microscopy (left: before experiments), resonant scanning confocal microscopy of live cells stained with plasma membrane dye (PM; middle), and immunofluorescence (IF) staining for endothelial junction protein VE-cadherin in fixed cells (right). Scale bars: 500 μm (phase contrast), 30 μm (PM, IF). (d) Representative 2-dimensional maximum intensity projection image (MIP) (left) and corresponding positions of bacteria (right: 25-75% interval boxplots) in the input channel from a 3D dataset captured under static conditions. -series were captured before experiments to measure input channel depth and calculate flow rates required to achieve shear stress of 1 dyn/cm² at the endothelial surface and confirm uniform distribution of bacteria.

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