Visualizing Bacterial Protein Distribution in Three Dimensions by Fluorescence Imaging

Begin with a sealed slide containing bacterial cells labeled with a cytoplasmic red fluorescent marker and a protein-specific green fluorescent marker.

Observe the slide under a fluorescence microscope.

Locate a field containing well-isolated bacterial cells and focus on the center of a representative cell.

Set the first fluorescence channel and acquire a z-stack image at specific parameters to capture the three-dimensional shape of the representative cell and its nearby cells.

Then, switch to the second fluorescence channel and acquire another z-stack image of the same cells with the same parameters.

It captures the distribution of bacterial proteins within the cells.

Crop individual cells from each z-stack to isolate them for single-cell analysis.

Align these images to visualize the distribution of bacterial proteins relative to cell shape, enabling precise analysis during host–pathogen interactions.

Immediately after sealing the cover slip, place the slide onto a microscope stage and, after five minutes of thermal equilibration, use the microscope focus wheels to bring the middle of a cell into focus. In the associated microscope software under ND Acquisition, check the Z box to acquire