The overall goal of this procedure is to monitor the zero DUM infection cycle using composite diffuse light imaging tomography with integrated micro CT to create a four D movie of the infection. This is accomplished by first preparing a bioluminescent bacterial inoculum. The second step of the procedure is to infect mice by oral gavage with zero dunum.
The infected mice are then image daily using D light micro ct, and the final steps are to reconstruct the 3D DLI micro CT data and compile it into a four D movie of the infection cycle. Ultimately, results can show the change in bacteria load distribution and localization through the four D multimodality imaging of infected mice. Dr.James Collins.
A post-doc in my lab will be demonstrating the procedure today In the late afternoon on the day before the infection set out a cryo vial of zero deum strain IC 180 to thaw as soon as it is thawed at one cryo bead of bacteria to 15 milliliters of lb. With can mycin then set the culture to grow overnight at 37 degrees Celsius with shaking at 220 RPM. Also culture 15 milliliters of un inoculated LB to control from medium contamination the next morning.
If the UN inoculated control is not turbid, transfer the inoculated culture to a 50 milliliter tube and centrifuge it at 4, 000 RPM for 10 minutes At four degrees Celsius, bring the pellet back up in a volume of PBS and repeat the centrifugation. Then thoroughly resuspend the cells in 1.5 milliliters of PBS to create the bacterial inoculum. To verify that the ICC 180 culture is bioluminescent and to estimate the number of bacteria, image the culture in the ivus spectrum CT using open filter bioluminescence imaging and use the auto setting in the living image, 4.3 0.1 wizard.
This is described in detail in a previously published video. To demonstrate the infection of mice with bioluminescent see rodent, we will infect one mouse and use one mouse for a mock infection with PBS. Prior to performing the infection, anesthetize the mouse with 3%isof fluorine using an XGI eight anesthesia system to provide humane restraint while the anesthesia takes hold thoroughly.
Mix the bacterial inoculum and draw a known number of bacteria in 0.2 milliliters into a syringe for oral gavage. Now retrieve the first mouse to infect by a firm grip on its scruff. Please note that mice were depleted prior to imaging using detory cream as described in the text article, push the needle towards the roof of the mouth over the tongue and down the esophagus and inject the bacterial inoculum into the stomach.
If there is resistance, do not force the needle because this will put the inoculum in the lungs instead, gently reinsert the needle. Subsequently, if the mouse has trouble with breathing or walking, it should be euthanized for the mock infection. Gavage a mouse with 0.2 milliliters of PBS to confirm that the oral gavage was performed correctly.
Image the mice in the IVUS spectrum CT using open filter bioluminescence imaging and use the auto setting in the living image. 4.3 0.1 imaging wizard as described previously, Delight micro CT involves delight optical imaging integrated with a low radiation dose micro CT scan. Because the dose accumulates the animals of each imaging session, we aim to keep the dose as low as reasonably possible.
Due to these concerns, the mice should be called at the first sign of detrimental symptoms or at the end of the micro CT imaging period. For this study, optimize the automatic exposure feature parameters in living image 4.3 0.1 for imaging bacterial luciferase in vivo. First select edit, then preferences, then acquisition and then auto exposure window.
Next, select range values followed by experiment time in seconds and set the maximum to 300 seconds. Finally, select the minimum target count followed by luminescence and set this value to 10, 000 counts. Once the system has been initialized, the anesthesia has been set up and the x-ray safety interlocks have been checked.
Proceed with anesthetizing a mouse. Use an XGI eight anesthesia system with an oxygen flow rate of two liters per minute and 3%isof fluorine. With the mouse ready for imaging, open the imaging wizard in the software.
This tool automatically optimizes several parameters to provide the best possible signal to noise ratio for each selected emission filter. When prompted by the imaging wizard, include the five sixty five eighty six hundred and six twenty nanometer emission filters. Then make sure the one mouse micro CT scan is selected and click acquire to start the imaging.
After returning the anesthetized mouse to its cage, remove the one mouse imaging platform from the spectrum CT and disinfect the animal platform using 1%tri gene.Note. If the animal platform is heavily soiled, the foam pad can be replaced. Continue imaging the mice daily up to eight days post-infection to generate the longitudinal imaging data.
Longer trial periods increase the likelihood of artifacts due to cumulative radiation exposure. To produce high quality reproducible three DD light micro CT reconstructions, a number of steps are required. First, open living image software, 4.3 0.1 and select Browse from the browse window.
Open the folder containing the delight micro CT files by clicking load. Open the DLI micro CT file in the living image browser. For example, see deum day seven post-infection.
In the tool palette, select surface topography followed by nude mouse. Adjust the threshold and then click generate surface. If the surface reconstruction is successful, a surface outline will be displayed in the 3D view window.
To view the rendered micro CT scan in the 3D view window, either hide the surface outline or decrease the surface opacity. Both options are accessed by first selecting 3D optical tools. Then either deselect display surface object or adjust the opacity slider bar to provide detailed anatomical localization of the three DD light reconstruction.
In reference to the animal skeleton, alter the 3D volumetric data. To view the skeleton with optimum contrast, click 3D multimodality tools and adjust the histogram slider to the right until only the bones are clearly visible. Then if not already selected, click logarithmic histogram, reverse coloring gradient illumination quality and de-noise.
If desired, crop the image to remove any unwanted structures from the 3D reconstruction from the tool palette, select D light 3D reconstruction. Make sure that only the five sixty five eighty six hundred and six twenty nanometer filters are selected. Then click start.
This opens the data preview window with the spectrally filtered bioluminescence signals from the mouse imaged in 2D. These signals are automatically threshold by the software, but can be adjusted manually if necessary, using tabs at the bottom of the menu. The threshold determines the minimum data intensity that will be included as data in the reconstruction.
Next from the tool palette, select the D light micro CT 3D reconstruction and check the optical properties. By clicking on the properties tab, the tissue properties should be set to mouse tissue, the source spectrum set to bacteria. Now click reconstruct to perform the D light reconstruction.
The actual location of the D light signal circled in red is within the pelvic girdle. This is easier to interpret when the image rotates during the movie. To generate the DLI micro CT 3D movie, go to the tools menu and select 3D animation preset animations, counterclockwise axis, and then total duration.
Set this value to 10 seconds or 25 frames per second. Once the settings for the 3D animation are correct, press record and save the DLI micro CT 3D reconstructions as a VI files. It is vital to make these reconstructions using identical settings so that they're comparable.
It's also important to make the videos using the same number of frames per second and total duration. Otherwise, the timing of the 40 video is not homogenous. At the computer, open Windows live movie maker and create a new file.
Insert the DLI micro CT reconstructions in chronological order from day one post-infection. Using the tools tab, add captions to the start of every video by selecting the video, selecting home, and then selecting add caption. The text in the caption can be formatted from the toolbar and the location of the caption is also adjustable.
Now add a title page by clicking home and then title. Type your title into the box using the toolbar to format the text. Next, save the project as an MMP file.
This is essential if you want to amend the movie. Finally, save the movie as a WMV file from the movie maker menu, select save movie, and choose the four computer option controls are essential to good technique. Prior to infecting mice, the bacterial inoculum used was determined to be bioluminescent.
After the oral gavage with bioluminescent see rodent, it was checked whether the signal could be observed in the stomach of the animal illustrated by a white arrowhead and absent from the lungs. The same mouse will be shown in subsequent examples of an individual infection. Zero denture is an extracellular pathogen confined to the intestinal lumen during the demonstrated infection.
It is thus shed in the feces, which were analyzed to show that colonization increases from day two to day six or seven when the infection peaks daily. D light micro CT was used to evaluate the spatial distribution of bioluminescent bacteria within this mouse using the skeleton as an anatomical reference. At day three, post-infection, small bioluminescent foci were observed in the colon illustrated by a blue arrow.
These foci exhibited a moderate increase in bioluminescence intensity by day five post-infection with little change in spatial distribution at day seven post-infection, there was a significant increase in bioluminescence and the bioluminescent foci spread across the entire colon. Three DD light micro CT reconstructions from days one to eight post-infection illustrate the spread of the sero deum infection. The bacterial infection begins as distinct foci within the proximal gastrointestinal.
By day three, the infection spreads to the colon. Over the next few days, the foci of infection spread further growing in size and number. The infection hits its peak on day seven.
Then on day eight, the colonies were reduced to two distinct bacterial foci in the proximal and distal colon. This technology will enable researchers to study mechanism for bacterial colonization and infection in real time and to investigate effectiveness of intervention strategies.
