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

Figure 2

Focal depth, sensitivity, and accuracy of imaging-based bacterial quantification in microfluidic devices. (a) Sample 2-dimensional maximum intensity projection image of -series showing B. burgdorferi transmigration through endothelia (red) after 1.5 h of flow (left),\ and isosurface-rendered bacteria identified in the input (yellow) and collection (blue) channels of the same -series by volumetric object counting. Scale bar: 100 μm. (b) Sensitivity comparison of imaging- and plate reader-based bacterial quantification methods. Orange line: background fluorescence intensity of perfusion buffer in plate reader. Inset: magnified view of region in upper graph indicated by dashed box. Fluorescence intensity values for bacteria counted in 3D imaging datasets were extrapolated from standard intensity vs. bacterial number curves from plate reader samples. (c, d) Precision and accuracy of bacterial counting by volumetric object identification in -series. In (c), the coefficient of variation (CV) for triplicate -series acquired at multiple locations in each microfluidic device was compared to CV for bacterial counts in Petroff-Hausser counting chambers (PHCC) used to measure input numbers of bacteria. NS: not significant (; two-tailed -test). independent microfluidic devices, 3 independent bacterial cultures (PHCC). (d) Numbers of bacteria counted by volumetric object identification in input channels under no-flow conditions before experiments (“actual”) compared to numbers of bacteria expected within each -stack based on input numbers calculated from PHCC measurements. Gray shading: CV of PHCC counts (i.e., expected input measurement variation). All figure . In (b), most error bars are too small to be visible.

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