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

Summary

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.

Abstract

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.

Introduction

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 biofilms^2,3. 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 organism^4,5. 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 infection^6,7. 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 bacteria^10,11. 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 implants^14,15 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 µm. 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 90° 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° 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 °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.

Protocol

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

1. Turn on PMT cooler, it typically takes ~15 min to reach the setpoint (e.g., 4 °C). Perform the rest of the initialization steps before turning on the PMTs.
2. 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.
3. 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° 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.
4. 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.
5. 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.
6. 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.
7. Open the enclosure door.

2. Optionally, perform a background scan with the X-ray off

1. Connect the polycapillary optics again to the X-ray source.
2. 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.
3. 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 µm and for a low-resolution scan, the step size will be 250 µm. 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.
4. 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.

3. Perform a sample scan with the X-ray on

1. Ensure the sample is still in the correct position with the laser cross head to begin the scan.
2. 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.
3. 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: For a high-resolution scan, the step size will be 1000 µm and for a low-resolution scan, the step size will be 250 µm. 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.
4. Obtain the scan for the sample with the X-ray on.
5. 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.
6. Turn off the PMT power supply prior to opening the door.

4. Forming the image

1. Validate the current scan is imaging the area of interest of the target. If not, stop the current scan by hitting the Stop button.
2. Adjust the scanning positions in the controlling software again and hit the Run button again.
   ​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.

5. Culturing bacteria for imaging in sterile conditions (If bacterial grown sensors are imaged)

1. 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).
2. Shake the bacterial culture gently at 37 °C for 16-18 h until the stationary phase.
3. 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.
4. 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.
5. Sterilize the Tryptic Soy Agar (TSA) by autoclaving and then cool by mixing until the temperature reaches 45 °C. Inoculate the bacteria into the TSA.
6. Pipette the diluted bacterial culture (100 µL) onto the surface of the implantable sensor.
   NOTE: Implants were sterilized by immersion in 70% ethanol for 5 min and stored in sterile PBS.
7. Pipette 100 µL of uninoculated TSA over another sterile implant as a control
8. Add an additional 100 µL of uninoculated TSA over the implantable sensor before it is incubated at 37 °C for 48 h prior to implantation.

Results

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

Discussion

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° angle to ...

Materials

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