The overall goal of the following experiment is to obtain a quantitative image of an infection in live animals that can allow localization of the infecting organism within the host tissues and organs. Anesthetized mice are first infected intratracheally with bioluminescent bacteria. Infected mice are then injected with Luciferian, which allows live imaging in the IVIS system to be carried out.
Once whole body images have been obtained, the infected lungs are harvested. Imaging of the isolated organs can be performed to demonstrate the true localization of the pathogen. Following imaging, the lungs are homogenized and dilution plated on bacterial growth medium for quantification of the microbial load.
Ultimately, results are obtained that show the location and number of pathogens present within the host in real time. The main advantage of this technique over existing method is that pathogens can be visualized and monitored in live animals by real time imaging providing insight into the spatial tics of microbial infection. Demonstrating this procedure will be Dr.Mihi Chang, a postdoctoral fellow in my laboratory, and SWAT Cillo, a researcher in my laboratory.
Start by intraperitoneal, injecting six to 12 week old valve C mice with a premixed anesthetic solution containing 100 micrograms of ketamine and 10 micrograms of xylazine per gram of mouse weight. Place the mice back in their cages until the anesthetic takes effect. To determine whether the anesthesia is complete, squeeze the foot pad of each mouse and ensure that no pedal reflex reaction occurs.
Once the animals are fully anesthetized, take one mouse and place it on the intubation. Stand lying on its back. Use tape to fix the mouse's legs and arms onto the stand.
Fix a rubber band to the intubation stand. Then place it under the mouse's upper incisors. Once the mouse is immobilized, use forceps to gently pull the tongue out of the mouth.
Next, gently insert the speculum with the otoscope inside the mouse's mouth and find the larynx opening, which is located in front of the esophagus on the ventral side of the oropharynx. Once the larynx opening has been identified, insert a 22 gauge one inch catheter containing a guide wire into the trachea until the catheter hub reaches the incisors. Remove the guide wire and connect the catheter to the plastic pump.
Then push air into the catheter. The mouse's chest will inflate if the intubation is correct. Once correct placement of the catheter has been confirmed, pipette 50 microliters of bioluminescent bacterial solution at the desired concentration into the catheter, and attach a one milliliter syringe with 50 microliters of air to push the solution into the mouse's lungs.
Two or three times, remove the catheter and place the mouse back in its cage to allow it to recover from the anesthesia. Before performing the bioluminescence imaging anesthetize the mice using isoflurane. Once the mice are fully anesthetized, remove them from the induction chamber and gently apply optical ointment to protect the animal's eyes.
During imaging, place the mice in the imaging chamber each and one of the nose cones on the anesthesia manifold. Use a light baffle between the mice to prevent the reflection of light between adjacent animal subjects. Finally, inject each mouse intraperitoneal with 150 micrograms per gram of body weight of Lucifer.
Allow the Lucifer to disperse throughout the animal's bodies for 10 minutes. Then begin imaging. Start the living imaging software and initialize the IVIS imaging system.
To set the parameters first, click on sequence setup. Then select the luminescence and photograph imaging mode. Select photographic luminescent and structural light images as part of the image sequence to enable DLIT 3D reconstruction.
Next, set the exposure time to one minute. Then adjust the binning to medium and the F-stop to one. Set the excitation filter to block and the emission filters to open for 3D reconstruction enable multiple filters of different wavelengths.
To allow accurate localization of the signal source, select the FOV corresponding to the number of mice being visualized. Once all the settings have been defined, click add in the image wizard in the acquisition control panel to add the sequence setup and click acquire to begin the imaging sequence during the acquisition. Record experimental details and conditions in the edit image window and save the images.
Finally, remove the mice from the imaging chamber and return them to their cages. Monitor animals constantly until they recover from anesthesia. For tissue imaging humanely euthanize the mice.
At this point, aseptically harvest the lungs of each mouse and transfer the organs to a sterile petri dish. Place the Petri dish containing the infected lungs inside the imaging chamber and acquire bioluminescence images in the same manner as described earlier in this video. Once images have been obtained, remove the Petri dish from the imaging chamber and using sterile forceps, transfer the lungs to a tube containing one milliliter of sterile PBS using a homogenizer homogenize the lungs for two to five minutes.
Prepare serial dilution of the lung homogenates in sterile PBS. Then plate 100 microliters of each dilution onto Petri dishes containing selective medium. Incubate the cultures at 37 degrees Celsius until bacterial colonies are clearly visible.
Then count the CFU to assess the level of infection. Start the living image software and open the desired image files from the file browser. In the tool palette.
Click on image adjust to modify the correction and filtering settings as well as the color scale thresholds. To quantify the luminescence intensity, use the region of interest or ROI tools from the pull down list, select ROI shape number and size. Then click on ROI measurement and save the quantitative data output to reconstruct 3D images.
First, load the image sequence, including the structured light images. Click on surface topography in the tool palette. Then in the pull down list, select the reconstruct tab to generate a surface topography.
Next, select DILT 3D reconstruction from the tool palette. Click on the properties tab in the pull down list and set the tissue properties and source spectrum to muscle. Click the analyze tab and select the wavelength for analysis.
Then click start. Once all the settings have been adjusted, click on reconstruct to initiate the 3D analysis. When the 3D reconstruction is complete, select 3D view to display the results by clicking on the results tab, photon density, data voxels, and algorithm parameters can be viewed, save the results and export the data and images for further analysis.
As shown in this figure, the two mice on the right which were intratracheally infected with 10 to the sixth CFU of bioluminescent bacteria produce a strong signal from the pulmonary area, whereas the uninfected mouse on the left does not. A similar analysis Using dissected lungs from infected mice alone confirms that the luminescence is emanating from the lungs rather than from some other closely juxtaposed tissue. The signal emanating from the sample can easily be quantified as total flux within a region of interest.
A tomography analysis confirms that the position of the luminescence displayed in these images as red boxes falls within the area containing the lungs of the mouse as determined by 3D reconstruction. Finally, the similarity between the measured photon density curve on the left and the simulated photon density curve on the right attest to the good quality of the reconstruction. After watching this video, you should have a good understanding of how to image animals infected with precedent and analyze the images for localization and quantification of reminiscence.
