Computational protocol: Second harmonic generation imaging of corneal stroma after infection by Pseudomonas aeruginosa

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Protocol publication

[…] Image stacks were reconstructed three-dimensionally in Imaris (Bitplane, Concord, MA). 3D movies were made using the Leica Application Suites built-in software (Leica, Heidelberg, Germany). Orientation of PA with respect to collagen lamellae was analysed using the Directionality plug-in in ImageJ (National Institutes of Health, Bethesda, MD). For ex vivo corneas, directionality assessment was performed on the full 1024X1024 image. For in vivo models, smaller 256 × 256 regions were used to isolate the specific banded regions of PA infection. Statistical analysis was performed in SigmaPlot (Version 12.5, Systat Software, San Jose, CA). Linear regression was used to determine the correlation between PA orientation and collagen. The correlation coefficient from three independent XY slices in each stack were calculated and are expressed as mean ± standard deviation. To compare differences between two groups (in the 6 hour condition), a t-test was used. For comparisons between three groups (in the 18 and 24 hour conditions), a One-way ANOVA with appropriate post-hoc comparison test was used. A One-way ANOVA was also used to compare differences in the overall correlation coefficients between time points. P < 0.05 was considered statistically significant. All experiments were repeated a minimum of two additional times. […]

Pipeline specifications

Software tools Imaris, ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Pseudomonas aeruginosa, Oryctolagus cuniculus, Bacteria