Imaging Bacterial Localization and Host Interaction in Human Lung Tissue

Take a fluorescently labeled Staphylococcus aureus suspension and human alveolar lung tissue infected with the same fluorescently labeled bacteria.

Insert the distal tip of the fibered confocal fluorescence microscopy probe into the bacterial suspension and scan.

Bright fluorescent signals confirm bacterial membrane labeling and successful probe activation

Record the fluorescence as the probe moves through the suspension.

Using a hydrogen peroxide-soaked lens clearing tissue, wipe the probe to remove residual bacteria and prevent background fluorescence

Bring the cleaned probe into direct contact with the tissue and scan the surface.

In the tissue, collagen and elastin fibers appear as an autofluorescent matrix, while fluorescently labeled bacteria are visible as distinct bright spots.

The alveolar spaces, lacking fluorescence, appear as dark regions.

Record a video to capture both tissue-adhered and loosely associated bacteria, enabling real-time visualization of bacterial infection in host alveolar lung tissue.

To begin data collection, click start on screen or press the left foot pedal to turn on the laser. Next, image each of the bacterial suspension samples. Insert the distal end of the FCFM imaging fiber and move the fiber slowly through the suspension to interrogate the sample.

Record videos of any length by pressing the right foot pedal or selecting the on-screen record controls as the fiber moves slowly around the sample. Clean the distal end of the FCFM imaging fiber with 8% hydrogen peroxide and lens cleaning tissues between samples. To image each of the lung tissue samples, insert the distal end of the FCFM imaging fiber into the sample ensuring that direct contact between the end of the fiber and the tissue is made.

Then gently move the end of the imaging fiber around to interrogate the sample. Lifting the end of the fiber away from the tissue will remove the tissue from the focal plane. However, this may be used to image labeled bacteria that are not adhered to the tissue.

Record videos of any length by pressing the right foot pedal or selecting the on-screen record controls as the fiber moves slowly around the sample.