Quantifying Polarity and Dynamics of a Secretion System Component in Legionella pneumophila

Begin with Legionella pneumophila, a respiratory pathogen expressing a fluorescently tagged secretion system component required for host infection.

To quantify the polarity of these components, acquire fluorescence images, and adjust contrast to distinguish individual cells.

Place rectangular regions from the cell pole toward the cytoplasm to measure fluorescence gradients.

Mask these regions to extract mean intensity and variance, then calculate polarity scores as variance-to-mean intensity ratios.

High polarity scores indicate targeted recruitment of components at the bacterial poles.

For dynamic measurements, capture two time-lapse images and adjust the contrast to visualize the cells clearly.

Place square regions at the cell center to measure cytosolic fluorescence.

Mask entire cells to correct for photobleaching and define background regions between cells for noise subtraction.

Export all intensities and calculate the change in fluorescence over time.

Increased intensity indicates clustering, while decreased intensity reflects the dynamic redistribution of secretion components within the bacteria.

To quantify polarity, adjust the image contrast so the bacteria are clearly visible.

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