This study outlines a method for tracking bacterial infections in vivo using bioluminescent bacteria in an anesthetized mouse model. The technique allows for real-time monitoring of infection progression through bioluminescence imaging.
Begin with an anesthetized mouse and, using a catheter, inject a suspension of pathogenic bacteria into the urinary bladder.
These bacteria carry a bioluminescent operon, which enables autonomous light production to track the infection.
The bladder’s inner lining is a specialized epithelium that separates the lumen from the underlying tissue.
The bacterial adhesion proteins bind to the glycoprotein receptors on the host epithelial surface, triggering receptor-mediated endocytosis.
The internalized bacteria replicate to form dense intracellular bacterial communities.
The bacteria express the bioluminescent operon, producing a luciferase enzyme and an enzyme complex that synthesizes the luciferase substrate.
Luciferase oxidizes the substrate to emit visible light.
Position the mouse inside the imaging chamber and capture the bioluminescent signal.
Select a region of interest and quantify the bioluminescence intensity.
Monitor this signal over time as a real-time indicator of bacterial load and infection progression.
Open the BLI acquisition software and click on Initialize in the imaging device to test the camera and stage controller system and to cool the charge-coupled device camera to minus 90 degrees Celsius. Click on Acquisition, AutoSave To. Select Luminescence and Photograph. Check the default luminescent settings by setting the excitation filter to Block and emission filter to Open.
Set the exposure time to auto when taking the first image. For in vivo measurements and bright signals, set the exposure time to around 30 seconds. Reduce the exposure time if a warning appears due to a saturated image.
Select the medium binning, F/stop one, and choose the correct FOV. Set the subject height to 1 centimeter when imaging mice. Image up to 5 mice simultaneously and separate the animals using the light baffle to prevent reflection.
Close the door and click on Acquire to start the imaging sequence. Then fill in detailed information about the experiment. Remove mice from the imaging chamber and return them to their cage. Check for full recovery after anesthesia. Then return the cages to the ventilated racks until the next imaging cycle.
Start the imaging software and load the experimental file by clicking on Browse. Use the tool palette to adjust the color scale of the image. Use the ROI tools to draw a region of interest on the image, ensuring that it is large enough to cover the complete area and using the same dimensions for all the images. Then, click on ROI measurement to quantify the light intensity.
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