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 a Z stack and click the Home button to set the middle of the cell as the starting point. Set the Step size to 0.1 micrometers and the Range to four micrometers, and make sure that the Z Device is set to the Piezo stage.

It is critical that there is a sufficient blurring on both the top and bottom of the cell so that the cell is almost indistinguishable from the background. Set the fluorescent channels under the lambda window to the settings for the fluorescent molecules being imaged, and confirm that the order of experiment is set to lambda so that a complete Z stack will be obtained in each color channel before switching. Then, click Run now to begin the image acquisition.

When all of the images have been acquired, open the files in an appropriate image analysis software program. Draw a box around an individual cell and duplicate that cell two times, once for each channel, making sure the Duplicate hyperstack box is checked, and change the channel to either one or two. Once both stacks are available, select Images, Stacks, Tools, and Concatenate to combine the images.