High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

This technique will allow us to image and map biochemical changes of implant-associated infections through bone and tissue. We can obtain high resolution images of the variations in chemical concentrations near implant surfaces. This will allow us to monitor the local chemical environment near implant-associated infections.

Demonstrating the bacterial culturing procedure will be Erin Levon, a graduate student from Department of Biological Sciences. To begin, turn on PMT cooler and perform the rest of the initialization steps before turning on the PMTs. Then open the imaging system controlling software and move the stage x-axis and y-axis to the desired starting position.

Place the sample on the movable xyz stage and position the sample height so the radio luminescent device is 5 to 5.5 centimeters below the poly capillary focus optics by raising or lowering the x-ray source and/or the stage. Also, position the specimen in the x-y plane with the help of the laser crosshead and remove the focusing optics for obtaining the plain radiograph of the sample. Secure the push button interlock at the front door of the imaging enclosure.

Then turn on the power for the x-ray source. Next, open the x-ray controlling software. Set the x-ray power and open the x-ray shutter with the x-ray controlling software.

Then open the software for the x-ray camera and hit the Exposure button to take the plain radiograph. Turn off the exposure and the x-ray, and then open the enclosure door. Connect the poly capillary optics again to the x-ray source.

Then close the enclosure, secure the interlock, and turn on the PMT power supply. Next, open the imaging system controlling software and specify the step size, scan speed, and scan area. Once all the parameters are set, start the scan by hitting the Run button.

Run a background scan with the x-ray off to determine the dark counts from any light present in the enclosure other than the sample. After ensuring that the sample is in the correct position with the laser crosshead, close the enclosure and secure the interlock. Then open the imaging system controlling software and enter the values for step size, scan speed, and scan area.

Once all the parameters are set, hit the Run button to start the scan and obtain the scan for the sample with the x-ray on. First, perform the low resolution scan with larger step sizes and higher scan speed to obtain a preliminary image of the target. After obtaining a low resolution scan of the desired area of the sample, obtain the higher resolution scan with a smaller step size and lower scan speed.

To prepare a fresh culture of staphylococcus aureus 1945, use one colony from a tryptic soy agar plate, streaked within one week to inoculate three milliliters of sterile tryptic soy broth. Then shake the bacterial culture gently at 37 degrees Celsius for 16 to 18 hours until the stationary phase. Next, pellet the culture from the tryptic soy broth via centrifugation at 4, 000 g for 10 minutes at room temperature and wash the pellet twice with PBS.

Quantify the bacterial concentration using optical density at 600 nanometer using the linear range, which is the optical density range where the Beer-Lambert law is verified. Then dilute the sample 200, 000 cells per milliliter using sterile PBS. Sterilize the tryptic soy agar by autoclaving, and then cool by mixing until the temperature reaches 45 degrees Celsius.

Next, inoculate the bacteria into the tryptic soy agar. Next, pipet the diluted bacterial culture onto the surface of the implantable sensor and 100 microliters of uninoculated tryptic soy agar over another sterile implant as control. Add an additional 100 microliters of uninoculated tryptic soy agar over the implantable sensor before it is incubated at 37 degrees Celsius for 48 hours prior to implantation.

After completing the scan, the images at 620 nanometer, 700 nanometer, and ratio respectively were generated in MatLab, and the change in color was indicative of the changes in the pH. As the basic pH region significantly absorbs emitted light than the acidic pH region, the lower pH region appears as a brighter signal at 620 nanometers. The scintillator emission at 700 nanometer functions as a spectral reference for inconsistencies in the scintillator film, tissue composition changes, and any changes that occur in the position of the detection optics from scan to scan.

The sample needs to be correctly positioned on the stage. PMT power supply should be turned off before turning on room light or opening the enclosure, and a low resolution scan should be performed prior to high resolution imaging. Instead of the x-ray source, we can use an ultrasound source to perform ultrasound luminescence chemical imaging for monitoring pH changes associated with implanted medical devices.

Yes, after optimization of the system, we were able to detect pH variations in the intramedullary cavity of the bone compared to the bone surface which could be used to study osteomyelitis.