Name: high spatial resolution chemical imaging of implant-associated infections with x-ray excited luminescence chemical imaging through tissue
Uploaded: 2022-09-30T15:00:00
Description: 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

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 p...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			Hudson, NH 03051</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Gadolinium Oxysulfide Europium doped (Gd2O2S:Eu) particles-~8.0 &#181;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&#8482;-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>Staphylococcus aureus subsp. aureus Rosenbach</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>&#160;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 ...

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.

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Chemistry

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