Visualizing Live and Dead Borrelia burgdorferi

Specimens were examined on a Zeiss AxioImager M2 microscope equipped with differential interference contrast (DIC) and epifluorescent illumination and were recorded with a Hamamatsu ORCA-R² C10600 camera. A cell proliferation assay was performed by direct counting using a bacterial counting chamber (Hausser Scientific Partnership, Horsham, PA, USA) and DIC microscopy. A SYBR Green I/PI assay was performed to assess the viability of B. burgdorferi, as previously described.^{13 (link)} The ratio of live (green) and dead (red) B. burgdorferi was calculated by counting the cells using a bacterial counting chamber and epifluorescence microscopy, as previously described.^{13 (link)} For the aggregated cells, three representative images of each sample were captured for quantitative analysis. Image Pro-Plus software was applied to select the green (including yellow) and red (including orange) areas of different morphological forms to calculate the integrated fluorescence intensity (equal to area × average density or average intensity) of the red and green portions, as previously described.¹⁶