The authors would like to thank Clemson University, COMSET and Clemson SC BioCRAFT. The XELCI setup was initially developed with funds from NSF CAREER CHE 12255535 and later by NIH NIAMS R01 AR070305-01.

This procedure follows the animal use protocols approved by the Clemson University Institutional Animal Care and Use Committee (IACUC). The experiments are carried out according to the Clemson University Biosafety Committee (IBC) and radiation safety committee (RSC) as well as following the relevant guidelines and regulations.
NOTE: A flow diagram of completing a XELCI scan is shown below in Figure 5 followed by a detailed step-by-step description of the imaging procedure.
1. Initialize the system and acquire a plain radiograph
<ol>
	<li>Turn on PMT cooler, it typically takes ~15 min to reach the setpoint (e.g., 4 &#176;C). Perform the rest of the initialization steps before turning on the PMTs.</li>
	<li>Open the imaging system controlling software. The controlling software program communicates and initializes the x-y-z axis motorized stage. Move the stage x-axis and y-axis to the desired starting position.</li>
	<li>Place the sample on the movable x-y-z stage. Position the sample height (z-axis), so the radioluminescent device is 5-5.5 cm below the polycapillary 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 (two red line-shaped laser pointers attached to the X-ray focusing capillary and positioned at 90&#176; to each other so that the lines intersect where the X-ray will focus). Turn off the lasers before X-rays and PMTs are turned on.</li>
	<li>Secure the push button interlock at the front door of the imaging enclosure. Turn on the power for the-ray source. Remove the focusing optics for obtaining the plain radiograph of the sample.</li>
	<li>Open the X-ray controlling software and set the X-ray power (by adjusting voltage and current). Open the X-ray shutter with the X-ray controlling software.</li>
	<li>Open the software for X-ray camera. Hit the exposure button to take the plain radiograph. Turn off the exposure and turn off the X-ray. 
	NOTE: If needed, move the sample to improve sample position or acquire a series of X-rays at different positions so they can be collated to get a larger radiograph view. One can also acquire a radiograph on a separate system, but co-registration between XELCI and radiography becomes more difficult if the specimen moves.</li>
	<li>Open the enclosure door.</li>
</ol>
2. Optionally, perform a background scan with the X-ray off
<ol>
	<li>Connect the polycapillary optics again to the X-ray source.</li>
	<li>Close the enclosure and secure the interlock. Turn on the PMT power supply. 
	NOTE: The PMT power supply should always be turned off whenever the door is open or about to be opened to avoid overexposure to light.</li>
	<li>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. 
	NOTE: For a high-resolution scan, the step size will be 1000 &#181;m and for a low-resolution scan, the step size will be 250 &#181;m. The scan speed can be chosen from 5 mm/s through the 1 mm/s. The area of the scan depends on the dimensions of the sample.</li>
	<li>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.</li>
</ol>
3. Perform a sample scan with the X-ray on
<ol>
	<li>Ensure the sample is still in the correct position with the laser cross head to begin the scan.</li>
	<li>Close the enclosure and secure the interlock. If the PMTs are off (e.g., turned off prior to opening the door), turn on the PMT power supply.</li>
	<li>Open the imaging system controlling software. 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. 
	NOTE:&#160;For a high-resolution scan, the step size will be 1000 &#181;m and for a low-resolution scan, the step size will be 250 &#181;m. The scan speed can be chosen from 5 mm/s through the 1 mm/s. The area of the scan depends on the dimensions of the sample.</li>
	<li>Obtain the scan for the sample with the X-ray on.</li>
	<li>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.</li>
	<li>Turn off the PMT power supply prior to opening the door.</li>
</ol>
4. Forming the image
<ol>
	<li>Validate the current scan is imaging the area of interest of the target. If not, stop the current scan by hitting the Stop button.</li>
	<li>Adjust the scanning positions in the controlling software again and hit the Run button again. 
	&#8203;NOTE: The y-axis is continuously recorded starting from the first row of the scan. While performing a scan, the number of counts per each wavelength and time since the last updated motor position is recorded. The time recorded will account for any changes in motor speed, thus the exposure time. For each pixel, counts/second is normalized. The y-axis motor travels to scan the end of the current row of the y-axis and the motor comes back to the starting position. Then the x-axis motor increases its position by a step size defined by the user and scans the second row of the y-axis. This process is cycled until the x-axis motor reaches the specified width for the x-direction. The user can control the scan size, motor speed, and motor starting positions. The step size will determine the size of the pixels in the final image of the y-axis.</li>
</ol>
5. Culturing bacteria for imaging in sterile conditions (If bacterial grown sensors are imaged)
<ol>
	<li>To prepare a fresh culture of Staphylococcus aureus 1945 (ATCC 25923), use one colony from a TSA (Tryptic soy agar) plate streaked within 1 week to inoculate 5 mL of sterile Tryptic Soy Broth (TSB).</li>
	<li>Shake the bacterial culture gently at 37 &#176;C for 16-18 h until the stationary phase.</li>
	<li>Next, pellet the culture from the TSB via centrifugation at 4000 x g for 10 min at room temperature (RT) and wash the pellet twice with Phosphate Buffer Solution (PBS) and resuspend the pellet in 5 mL of sterile PBS.</li>
	<li>Quantify the bacterial concentration using optical density at 600 nm using the linear range, which is the OD range where the Beer-Lambert law is verified (OD = kN; k is a coefficient relative to the molecular extinction and the length of the optical path, N is the bacterial concentration)16. Then dilute the sample to 105 cells/mL using sterile PBS.</li>
	<li>Sterilize the Tryptic Soy Agar (TSA) by autoclaving and then cool by mixing until the temperature reaches 45 &#176;C. Inoculate the bacteria into the TSA.</li>
	<li>Pipette the diluted bacterial culture (100 &#181;L) onto the surface of the implantable sensor. 
	NOTE: Implants were sterilized by immersion in 70% ethanol for 5 min and stored in sterile PBS.</li>
	<li>Pipette 100 &#181;L of uninoculated TSA over another sterile implant as a control</li>
	<li>Add an additional 100 &#181;L of uninoculated TSA over the implantable sensor before it is incubated at 37 &#176;C for 48 h prior to implantation.</li>
</ol>

<ol>
	<li>Arciola, C. R., Alvi, F. I., An, Y. H., Campoccia, D., Montanaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implant+infection+and+infection+resistant+materials:+A+mini+review.">Implant infection and infection resistant materials: A mini review.</a> International Journal of Artificial Organs. 28 (11), 1119-1125 (2005).</li><li>Renick, P., Tang, L., Li, B., Moriarty, T. F., Webster, T., Xing, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Device-related+infections.">Device-related infections.</a> Racing for the Surface. , 171-188 (2020).</li><li>Stewart, P. S., Franklin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+heterogeneity+in+biofilms.">Physiological heterogeneity in biofilms.</a> Nature Reviews Microbiology. 6 (3), 199-210 (2008).</li><li>Metsemakers, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture-related+infection:+A+consensus+on+definition+from+an+international+expert+group.">Fracture-related infection: A consensus on definition from an international expert group.</a> Injury. 49 (3), 505-510 (2018).</li><li>Arciola, C. R., Campoccia, D., Montanaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implant+infections:+Adhesion,+biofilm+formation+and+immune+evasion.">Implant infections: Adhesion, biofilm formation and immune evasion.</a> Nature Reviews Microbiology. 16 (7), 397-409 (2018).</li><li>Polvoy, I., Flavell, R. R., Rosenberg, O. S., Ohliger, M. A., Wilson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Imaging+of+Bacterial+Infection:+The+State+of+the+Art+and+Future+Directions.">Nuclear Imaging of Bacterial Infection: The State of the Art and Future Directions.</a> Journal of Nuclear Medicine. 61 (12), 1708-1716 (2020).</li><li>vander Bruggen, W., Bleeker-Rovers, C. P., Boerman, O. C., Gotthardt, M., Oyen, W. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+and+SPECT+in+Osteomyelitis+and+Prosthetic+Bone+and+Joint+Infections:+A+Systematic+Review.">PET and SPECT in Osteomyelitis and Prosthetic Bone and Joint Infections: A Systematic Review.</a> Seminars in Nuclear Medicine. 40 (1), 3-15 (2010).</li><li>Trampuz, A., Zimmerli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+Treatment+of+infections+associated+with+fracture-fixation+devices.">Diagnosis and Treatment of infections associated with fracture-fixation devices.</a> Injury. 37 (2), 59-66 (2006).</li><li>Ady, J., Fong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+for+infection:+From+visualization+of+inflammation+to+visualization+of+microbes.">Imaging for infection: From visualization of inflammation to visualization of microbes.</a> Surgical Infections. 15 (6), 700-707 (2014).</li><li>Truong-Bolduc, Q. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+the+NorB+Efflux+pump+in+the+adaptation+of+Staphylococcus+Aureus+to+growth+at+acid+pH+and+in+resistance+to+Moxifloxacin.">Implication of the NorB Efflux pump in the adaptation of Staphylococcus Aureus to growth at acid pH and in resistance to Moxifloxacin.</a> Antimicrobial Agents and Chemotherapy. 55 (7), 3214-3219 (2011).</li><li>Hengge-Aronis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+and+regulatory+mechanisms+involved+in+control+of+the+&#963;S+(RpoS)+subunit+of+RNA+polymerase.">Signal transduction and regulatory mechanisms involved in control of the &#963;S (RpoS) subunit of RNA polymerase.</a> Microbiology and Molecular Biology Reviews. 66 (3), 373-395 (2002).</li><li>Gao, R., Yan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phosphors+for+x-ray+detection.">Molecular phosphors for x-ray detection.</a> Science Bulletin. 67 (10), 1015-1017 (2022).</li><li>Gao, R., Fang, X., Yan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+stimuli-responsive+luminescent+films.">Recent developments in stimuli-responsive luminescent films.</a> Journal of Materials Chemistry C. 7 (12), 3399-3412 (2019).</li><li>Uzair, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformal+coating+of+orthopedic+plates+with+x-ray+scintillators+and+ph+indicators+for+x-ray+excited+luminescence+chemical+imaging+through+tissue.">Conformal coating of orthopedic plates with x-ray scintillators and ph indicators for x-ray excited luminescence chemical imaging through tissue.</a> ACS Applied Materials &amp; Interfaces. 12 (47), 52343-52353 (2020).</li><li>Uzair, U., Benza, D., Behrend, C. J., Anker, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasively+imaging+pH+at+the+surface+of+implanted+orthopedic+devices+with+x-ray+excited+luminescence+chemical+imaging.">Noninvasively imaging pH at the surface of implanted orthopedic devices with x-ray excited luminescence chemical imaging.</a> ACS Sensors. 4 (9), 2367-2374 (2019).</li><li>Begot, C., Desnier, I., Daudin, J. D., Labadie, J. C., Lebert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+calculating+growth+parameters+by+optical+density+measurements.">Recommendations for calculating growth parameters by optical density measurements.</a> Journal of Microbiological Methods. 25 (3), 225-232 (1996).</li><li>Giering, K., Minet, O., Lamprecht, I., M&#252;ller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+thermal+properties+of+biological+tissues.">Review of thermal properties of biological tissues.</a> Proceedings of SPIE - The International Society for Optical Engineering. , 45-65 (1995).</li><li>Hunter, R. C., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+pH-sensitive+fluoroprobe+(C-SNARF-4)+for+pH+microenvironment+analysis+in+Pseudomonas+aeruginosa+biofilms.">Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms.</a> Applied and Environmental Microbiology. 71 (5), 2501-2510 (2005).</li><li>Pogue, B. W., Leblond, F., Krishnaswamy, V., Paulsen, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiologic+and+Near-Infrared/Optical+Spectroscopic+Imaging:+Where+Is+the+Synergy.">Radiologic and Near-Infrared/Optical Spectroscopic Imaging: Where Is the Synergy.</a> American Journal of Roentgenology. 195 (2), 321-332 (2010).</li><li>Ryan, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+X-ray-excited+emissions+from+quantum+dots+and+biological+tissue+in+whole+mouse.">Imaging of X-ray-excited emissions from quantum dots and biological tissue in whole mouse.</a> Scientific Reports. 9 (1), 19223 (2019).</li><li>Yang, Y., Wang, K. -. Z., Yan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultralong+persistent+room+temperature+phosphorescence+of+metal+coordination+polymers+exhibiting+reversible+pH-responsive+emission.">Ultralong persistent room temperature phosphorescence of metal coordination polymers exhibiting reversible pH-responsive emission.</a> ACS Applied Materials &amp; Interfaces. 8 (24), 15489-15496 (2016).</li><li>Chen, H., Rogalski, M. M., Anker, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+functional+X-ray+imaging+techniques+and+contrast+agents.">Advances in functional X-ray imaging techniques and contrast agents.</a> Physical Chemistry Chemical Physics. 14 (39), 13469 (2012).</li><li>Allan, V. J. M., Macaskie, L. E., Callow, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+pH+gradient+within+a+biofilm+is+dependent+upon+the+limiting+nutrient.">Development of a pH gradient within a biofilm is dependent upon the limiting nutrient.</a> Biotechnology letters. 21 (5), 407-413 (1999).</li><li>Wang, Z., Deng, H., Chen, L., Xiao, Y., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+measurements+of+dissolved+oxygen,+pH+and+redox+potential+of+biocathode+microenvironments+using+microelectrodes.">In situ measurements of dissolved oxygen, pH and redox potential of biocathode microenvironments using microelectrodes.</a> Bioresource Technology. 132, 387-390 (2013).</li><li>Xiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+probing+the+effect+of+potentials+on+the+microenvironment+of+heterotrophic+denitrification+biofilm+with+microelectrodes.">In situ probing the effect of potentials on the microenvironment of heterotrophic denitrification biofilm with microelectrodes.</a> Chemosphere. 93 (7), 1295-1130 (2013).</li><li>Nakata, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+to+design+latent+ratiometric+fluorescent+pH+probes+Based+on+self-assembled+SNARF+derivatives.">A novel strategy to design latent ratiometric fluorescent pH probes Based on self-assembled SNARF derivatives.</a> RSC Adv. 2014 (4), 348-357 (2014).</li><li>Villano, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+multislice+sequence+for+3D+MRI-CEST+pH.">A fast multislice sequence for 3D MRI-CEST pH.</a> Magnetic Resonance in Medicine. 85 (3), 1335-1349 (2021).</li></ol>

The authors claim no conflicts of interest.

To be able to detect and study orthopedic implant-associated infections early to avoid complications from osteomyelitis and secondary surgical procedures, we have introduced XELCI as a novel, functional imaging technique. It is comparable with the currently available techniques for pH monitoring through tissue.
While positioning the sample for imaging, we use a laser cross head connected to polycapillary focusing optics with two intersecting line-shaped laser pointers at the 90&#176; angle to ...

In the United States, about 2 million fracture fixation devices are inserted annually, and 5%-10% of them lead to implant-associated infections1. These infections are harder to treat with antibiotics at later stages due to the heterogeneity and antibiotic-resistant nature of the biofilms2,3. If they are diagnosed early, infections can be treated with antibiotics and surgical debridement to prevent extra medical costs for a second surgery to replace hardware at the treated fracture site. Plain radiography and other advanced radiographic techniques are applied in the diagnosis of orthopedic implant-associated infections, non-unions, and related complications. Although these techniques are used frequently to acquire structural information of the surrounding bone and tissue at the orthopedic implant, they are unable to provide biochemical information in the specific environment. Thus, we developed a novel X-ray excited luminescence chemical imaging (XELCI) technique for high-resolution imaging of biochemical information noninvasively at the implant site. Diagnosis of orthopedic implant-associated infections is commonly carried out by one or a combination of different means. Clinical observations (pain, swelling, redness, wound discharge, etc.) suggest the first signs of infection. Later, radiological and laboratory experiments are carried out to confirm the failure of bone healing progression and identify the pathogenic organism4,5. Nuclear medicinal techniques such as computerized tomography (CT), magnetic resonance imaging (MRI), and radionucleotide methods such as Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) are in use for better visualization of the infected implant and the associated infection6,7. CT and MRI are advantageous in determining bone necrosis and soft tissue abnormalities, respectively but cause interferences at a close distance to the metal implants8. Different X-ray methodologies such as SPECT and PET in combination with radioisotope-labeled analytes as in vivo imaging contrast agents are widely utilized to diagnose implant-associated osteomyelitis2. Current applications combine both data from CT scanning and labeling data from either SPECT or PET to generate anatomical information9. Although one or more of these imaging modalities are used to aid infection diagnosis, they cannot detect the pH variations associated with infection early to initiate the treatments with antibiotics to avoid extra medical and surgical expenses.
The main advantage of utilizing the imaging system used in this study for monitoring implant-associated infections is its ability to reveal biochemical information about the biofilm microenvironment with a spectral reference. Although the main focus is on imaging and mapping pH at the infected site, this method can be altered to monitor other biomarkers specific to implant-associated infections. Thus, XELCI allows understanding the pathophysiology of the infection. The high spatial resolution imaging allows mapping heterogeneity as the infection grows. pH at the surface where the biofilm formation occurs is very important to understanding biochemical changes. Also, other microenvironment changes can occur due to antibiotic-related stress responses by bacteria10,11. Due to surface-specific and high spatial resolution imaging, the antibiotic effect on the biofilm microenvironment can be monitored. The technique can also be used to study the biofilm environment for targeted drug delivery experiments. We can study targeted low pH drug release or raising pH to make them more susceptible to work at higher pH.
Three specific characteristics of this imaging technique are X-ray resolution, Implant surface specificity, and chemical sensitivity (Figure 1A). These characteristics can be compared with the currently available imaging techniques for imaging orthopedic implant-related infections (Figure 1B). Once irradiated with X-rays, phosphor particles coated on the implant surface generate red and near-IR (NIR) light that can penetrate through a few centimeters of tissue (albeit with some attenuation)12,13. Table 1 shows some of the features of the developed imaging system compared to other ways that have been used to measure pH in biofilms or through tissue.
XELCI is a novel imaging technique to acquire high spatial resolution chemical information optically near implanted medical devices in combination with X-ray excitation, as shown in Figure 2. Here the selective excitation and optical detection of X-ray excitable phosphor particles is utilized. The implant is coated with two layers, a pH-sensitive dye incorporated polymer layer over a layer of scintillator particles. Once a sequence of focused X-ray beams irradiates the implant, the scintillator layer generates visible light (620 nm and 700 nm). This produced light passes through the pH-sensitive layer modulating the luminescence spectrum depending on the pH of the surrounding environment. Low pH is generally associated with infection and biofilm formation; as the infection progresses, the pH changes from physiological pH (pH 7.2) to acidic (less than pH 7), and the pH dye in the sensor changes color and thus absorbance. The variation of the luminescence spectrum is shown in Figure 2E for Bromocresol green pH dye at pH 7 and pH 4. The transmitted light through tissue and bone is collected and the spectral ratio determines pH. To generate a pH image, the focused X-ray beam irradiates a point at a time in the scintillator film and scans the beam point-by-point across the sample. Previously, this technique was applied to image pH variation on the surface of the orthopedic implants14,15 and have tested it to monitor pH variations in the intramedullary canal through bone and tissue.
Figure 3 below shows a schematic of the imaging system. Basic components of the imaging system are the X-ray excitation source with poly capillary optics, a one-piece acrylic light guide connecting to two photomultiplier tubes, the x, y, and z motorized stage (30 cm x 15 cm x 6 cm travel) and the computer connected for data acquisition. The X-ray source, x,y,z stage, and collection optics (elbow, light guide, photomultiplier tubes (PMTs)) are in the X-ray proof enclosure, while the X-ray controller, power source for PMTs, function generator connected to the data acquisition (DAQ) board and computer are kept outside. A push-button, normally open switch, placed between the enclosure and the front of the door serves as an interlock. If the door is not fully closed (the interlock switch is open), the X-ray source will not turn on, and it will automatically turn off the X-ray source if it is opened during operation. The motors can execute a continuous scan as well as they can be moved to any discrete location. The scan speed for y-axis is usually 1-5 mm/s, while the step size on the x-axis can be chosen typically from 150-2000 &#181;m. The parameters can be chosen depending on the required spatial resolution. Even exposure times are confirmed by consistent speed throughout a continuous scan.
Once the focused X-ray beam is irradiated on the X-ray luminescence particles, the generated light will pass through the pH-sensitive film by modulating the light depending on the surrounding pH. The transmitted light will interact (scatter and absorb partially) with a tissue, while the light attenuation by scattering and absorption will increase as the tissue thickness increases. The collection optics includes a one-piece bifurcated acrylic light guide fitted with a reflective aluminum elbow (with a&#160;90&#176; bend and polished reflective interior surface) at the beginning. This is to ensure the light is collimated as soon as light reaches the light guide. These additions significantly improved the light collection efficiency. For further details, Figure 4 shows the machine drawings of the elbow and light guide. The 90&#176; elbow was machined out of aluminum with the internal surface polished to a mirror finish and the light guide was machined with Acrylic. We have also attached a broad range long-pass blue light filter (blocking 350-450 nm light) at the beginning of the elbow to ensure that only red light will pass through. The end of the one-piece acrylic light guide bifurcates into two streams leading to two different PMTs. The PMTs are enclosed in a small light-tight metal box that is in contact with a thermoelectric cooler to cool down the PMTs to ~5 &#176;C. At the beginning of one of the PMTs, a narrow range long-pass filter (blocking 570-640 nm light and passing 640-740 nm light) is attached to measure only the 700 nm light. Therefore, the 620 nm and 700 nm light can be calculated separately. The PMTs are set up in photon counting mode, and they generate transistor-transistor logic (TTL) pulses for each photon detected. A DAQ system counts the pulses (saturation point 20 million pulses per second) using USB communication. Two separate intensity maps are generated after processing the data, and a final image is created by considering the ratio of the signal wavelength intensity (620 nm) to the reference wavelength intensity (700 nm). This ratio accounts for differences in total light collection efficiency, which depend strongly on the position of collection optics, X-ray irradiation intensity, and tissue thickness. In addition, a spatially separated reference region without any pH indicator dye accounts for spectral distortion from wavelength-dependent tissue penetration. A graphics-based programming language is used for controlling the imaging system, and a basic flow chart of the operation is shown below. The imaging setup, except for the computer, X-ray controller, and DAQ unit, is enclosed in a safe X-ray enclosure to minimize radiation exposure.

<table><tbody><tr><td>90 degree elbow</td><td>Produced in Hilltop Technology Laboratory, 51 Parker, Irvine,CA</td><td></td><td></td></tr><tr><td>Bromo Cresol Green</td><td>Sigma-Aldrich</td><td>45ZW10</td><td></td></tr><tr><td>Bromo Thymol Blue</td><td>Sigma</td><td>76-59-5</td><td></td></tr><tr><td>ElectraCOOL Advanced thermoelectric cool plate</td><td>Pollock industries, White River, VT, USA</td><td>TCP 50</td><td></td></tr><tr><td>Ethanol</td><td>Beantown Chemical, 9 Sagamore Park Road&lt;br/&gt; Hudson, NH 03051</td><td>64-17-5</td><td></td></tr><tr><td>Gadolinium Oxysulfide Europium doped (Gd2O2S:Eu) particles-~8.0 &amp;micro;m</td><td>Phosphor Technologies Inc., Stevenage, England</td><td>UKL63/N-R1</td><td></td></tr><tr><td>LabVIEW</td><td>National Instruments, Austin, TX</td><td></td><td></td></tr><tr><td>Motorized Linear Vertical Stage Model (for Z axis)</td><td>Motion Control, Smithtown, NY, USA</td><td>AT10-60</td><td></td></tr><tr><td>National instruments c-DAQ 9171</td><td>National Instruments, Austin, TX</td><td>NI cDAQ&amp;trade;-9171</td><td></td></tr><tr><td>One piece acrylic light guide</td><td>Produced in Hilltop Technology Laboratory, 51 Parker, Irvine,CA</td><td></td><td></td></tr><tr><td>pH 4 buffer</td><td>VWR BDH Chemicals</td><td>BDH5024</td><td></td></tr><tr><td>pH 8 buffer</td><td>VWR BDH Chemicals</td><td>BDH5060</td><td></td></tr><tr><td>Phosphate Buffer Solution</td><td>MP Biomedicals, Irvine, CA. USA</td><td>2810305</td><td></td></tr><tr><td>Photo multiplier tubes Model P25PC-16</td><td>SensTech, Surrey, UK</td><td>Model P25PC-16</td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus &lt;/em&gt;subsp. &lt;em&gt;aureus Rosenbach&lt;/em&gt;</td><td>American Type Culture Collection (ATCC), Manassas, VA</td><td>ATCC 25923</td><td></td></tr><tr><td>Tryptic Soy Agar</td><td>Teknova, Hollister, CA, USA</td><td>&amp;nbsp;T0520</td><td></td></tr><tr><td>Tryptic Soy Broth</td><td>EMD Millipore, Burlington, MA, USA</td><td>1005255000</td><td></td></tr><tr><td>X-ray source-iMOXS</td><td>Institute for Scientific Instruments GmbH, Berlin, Germany</td><td></td><td></td></tr><tr><td>X,Y motorized stage-30 cm x 15 cm x 6 cm travel</td><td>Thorlabs Inc., Newton, NJ, USA</td><td>LTS300 and LTS150</td><td></td></tr></tbody></table>

high spatial resolution chemical imaging of implant-associated infections with x-ray excited luminescence chemical imaging through tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

As a preliminary study, we imaged the intramedullary rod sensor in a reamed tibia of a rabbit cadaver14. The sensor has three distinct regions: the reference region, pH 8 region (basic pH), and pH 4 region (acidic pH). The reference region is the scintillator (Gd2O2S:Eu) particle incorporated in roughened epoxy film. The distinctive acidic and basic pH regions represent infected and non-infected situations inside the intramedullary canal (Figure 6A</stron...

Watch this Scientific Journal Video about High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue at JoVE.com

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

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

high-spatial-resolution-chemical-imaging-implant-associated

Research

JoVE Journal

Chemistry

1.3K Views.  Clemson University. Deze techniek zal ons in staat stellen om biochemische veranderingen van implantaat-geassocieerde infecties via bot en weefsel in beeld te brengen en in kaart te brengen. We kunnen beelden met een hoge resolutie verkrijgen van de variaties in chemische concentraties in de buurt van implantaatoppervlakken. Dit zal ons in staat stellen om de lokale chemische omgeving in de buurt van implantaat-geassocieerde infecties te monitoren.De bacteriële kweekprocedure wordt gedemonstreerd door Er...

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

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Immunology and Infection

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medicine

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Bioengineering

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described ...

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

Samenvatting

Meer video's verkennen