Spatial Resolution for X-ray Excited Luminescence Chemical Imaging (XELCI).

Measuring chemical concentrations at the surface of implanted medical devices is important for elucidating the local biochemical environment, especially during implant infection. Although chemical indicator dyes enable chemical measurements in vitro, they are usually ineffective when measuring through tissue because the background obscures the dye signal and scattering dramatically reduces the spatial resolution. X-ray excited luminescent chemical imaging (XELCI) is a recent imaging modality which overcomes these limitations using a focused X-ray beam to excite a small spot of red light on scintillator-coated medical implants with well-defined location (because X-rays are minimally scattered) and low background. A spectrochemical indicator film placed over the scintillator layer, e.g., a polymer film containing pH-indicator dyes, absorbs some of the luminescence according to the local chemical environment, and this absorption is then detected by measuring the light intensity/spectrum passing through the tissue. A focused X-ray beam is used to scan point-by-point with a spatial resolution mainly limited by the X-ray beam width with minimum increase from X-ray absorption and scattering in the tissue. X-ray resolution, implant surface specificity, and chemical sensitivity are the three key features of XELCI. Here, we study spatial resolution using optically absorptive targets. For imaging a series of lines, the 20-80% knife-edge resolution was ∼285 (±15) µm with no tissue and 475 ± 18 and 520 ± 34 µm, respectively, through 5 and 10 mm thick tissue. Thus, doubling the tissue depth did not appreciably change the spatial resolution recorded through the tissue. This shows the promise of XELCI for submillimeter chemical imaging through tissue.

Upconversion Spectral Rulers for Transcutaneous Displacement Measurements.

We describe a method to measure micron to millimeter displacement through tissue using an upconversion spectral ruler. Measuring stiffness (displacement under load) in muscles, bones, ligaments, and tendons is important for studying and monitoring healing of injuries. Optical displacement measurements are useful because they are sensitive and noninvasive. Optical measurements through tissue must use spectral rather than imaging approaches because optical scattering in the tissue blurs the image with a point spread function typically around the depth of the tissue. Additionally, the optical measurement should have low background and minimal intensity dependence. Previously, we demonstrated a spectral encoder using either X-ray luminescence or fluorescence, but the X-ray luminescence required an expensive X-ray source and used ionizing radiation, while the fluorescence sensor suffered from interference from autofluorescence. Here, we used upconversion, which can be provided with a simple fiber-coupled spectrometer with essentially autofluorescence-free signals. The upconversion phosphors provide a low background signal, and the use of closely spaced spectral peaks minimizes spectral distortion from the tissue. The small displacement noise level (precision) through tissue was 2 µm when using a microscope-coupled spectrometer to collect light. We also showed proof of principle for measuring strain on a tendon mimic. The approach provides a simple method to study biomechanics using implantable sensors.

Impressively Printing Patterns of Gold and Silver Nanoparticles.

The optical and chemical properties of gold and silver nanoparticles make them useful for many applications, including surface enhanced spectroscopy-based biosensors, photostable colorants, enhanced photovoltaics, and nanoscale optical elements. We report a simple technique to generate patterns of gold and silver nanoparticles with controlled shape and shape-dependent optical properties using metal stamps to impress them onto a glass substrate or flexible polymers. The pressure flattens the nanoparticles, converting initially spherical nanoparticles into discs with reduced height and increased diameter. This deformation causes their localized surface plasmon resonance wavelength to red-shift. Nanoparticles were characterized by electron microscopy, atomic force microscopy, and dark field optical scattering spectroscopy. The deformed nanoparticle patterns had a lateral resolution limited by the nanoparticle diameter (single particles are partly flattened only where they contact the stamp). The method also (i) transfers the stamp's topography, with smooth stamps generating flattened nanoparticles with uniform height, and small changes in stamp height are evident in the nanoparticle height and scattering wavelength, and (ii) allows facile removal of undeformed nanoparticles using scotch tape, and patterns of deformed nanoparticles can be transferred to a thin polymer-film. The patterning process is simple and inexpensive. It can be performed by hand for demonstrations or artistic applications, with controlled force for plasmonics research, and potentially automated on reel-to-reel presses for large scale production.

Conformal Coating of Orthopedic Plates with X-ray Scintillators and pH Indicators for X-ray Excited Luminescence Chemical Imaging through Tissue.

We describe a pH-indicating material that can be directly implanted or coated on orthopedic implant surfaces to provide high-spatial-resolution pH mapping through tissue by X-ray excited luminescence chemical imaging (XELCI). This is especially useful for detecting local pH changes during treatment of implant-associated infections. The material has two layers: an X-ray scintillator layer with Gd2O2S:Eu in epoxy, which emits 620 and 700 nm light when irradiated with X-rays, and a pH indicator dye layer, which absorbs some of the 620 nm light in a pH-dependent fashion. To acquire each pixel in the image, a focused X-ray beam irradiates a small region of scintillators and the ratio of 620 to 700 nm light is acquired through the tissue. Scanning the X-ray beam across the implant surface generates high-spatial-resolution chemical measurements. Two associated challenges are (1) to make robust sensors that can be implanted in tissue to measure local chemical concentrations specifically for metal orthopedic implants and (2) to conformally coat the implant surface with scintillators and pH indicator dyes in order to make measurements over a large area. Previously, we have physically pressed or glued a pH-sensitive hydrogel sensor onto the surface of an implant, but this is impractical for imaging over large irregular device areas such as an orthopedic plate with holes and edges. Herein, we describe a chemically sensitive and biocompatible XELCI sensor material that can conformally coat the implant surface. A two-part commercial-grade epoxy resin was mixed with Gd2O2S:Eu and adhered to the titanium surface. Sugar and salt particles were added to the surface of the epoxy as it cured to create a roughened surface and increase the surface area. On this roughened surface, a secondary layer of diacrylated polyethylene glycol (PEG) hydrogel, containing a pH sensitive dye, was polymerized. This combination of epoxy-PEG layers was found to adhere well to the metal implant unlike other previously tested polymer surfaces, which delaminated when exposed to water or humidity. The focused X-ray beam enabled 0.5 mm spatial resolution through 1 cm-thick tissue. The pH sensor-coated orthopedic plate was imaged with XELCI, through tissue, with different pH levels to acquire a calibration curve. The plates were also imaged through tissue, with a low pH region on one section due to growth of a Staphylococcus aureus biofilm. A pH sensor-coated stainless-steel rod with two distinct pH regions was inserted in a rabbit tibia specimen, and the pH was imaged through both bone and soft tissue. These studies demonstrate the use of pH sensor-coated orthopedic plates and rods for mapping the local pH through tissue during biofilm formation by XELCI.

Luminescent Spectral Rulers for Noninvasive Displacement Measurement through Tissue.

A luminescent spectral ruler was developed to measure micrometer to millimeter displacements through tissue. The spectral ruler has two components: a luminescent encoder patterned with alternating stripes of two spectrally distinct luminescent materials and an analyzer mask with periodic transparent windows the same width as the encoder stripes. The analyzer mask is placed over the encoder and held so that only one type of luminescent stripe is visible through the window; sliding the analyzer over the encoder modulates the luminescence spectrum acquired through the analyzer windows, enabling detection of small displacements without imaging. We prepared two types of spectral rulers, one with a fluorescent encoder and a second with an X-ray excited optical luminescent (XEOL) encoder. The fluorescent ruler used two types of quantum dots to form stripes that were excited with 633 nm light and emitted at 645 and 680 nm, respectively. Each ruler type was covered with chicken breast tissue to simulate implantation. The XEOL ruler generated a strong signal with negligible tissue autofluorescence but used ionizing radiation, while the fluorescence ruler used non-ionizing red light excitation but required spectral fitting to account for tissue autofluorescence. The precision for both types of luminescent spectral rulers (with 1 mm wide analyzer windows, and measured through 6 mm of tissue) was <2 µm, mostly limited by shot noise. The approach enabled high micrometer to millimeter displacement measurements through tissue and has applications in biomechanical and mechanochemical measurements (e.g., tracking postsurgical bone healing and implant-associated infection).

Noninvasively Imaging pH at the Surface of Implanted Orthopedic Devices with X-ray Excited Luminescence Chemical Imaging.

Implanted medical device-associated infections are a leading cause of fixation failure, and early diagnosis is the key to successful treatment. During infection, acidosis near the implant plays a role in antibiotic resistance and low pH is a potential infection indicator. Herein, we describe a pH sensor which attaches to the implants to noninvasively image local pH with high spatial resolution. The sensor has two layers: a scintillator layer which emits 620 and 700 nm light upon X-ray irradiation and a pH indicator layer containing bromocresol green dye that absorbs 620 nm luminescence in neutral/basic pH and passes 700 nm light at all pHs. We also developed a dedicated imaging system capable of scanning relatively large specimens through thick tissues. A focused X-ray beam irradiates one spot on the sensor, and the 620 to 700 nm peak ratio is measured to determine the local pH; images are acquired by scanning the X-ray beam across the surface and measuring the pH point-by-point. The sensor was covered with varying thickness slices of chicken breast tissue (0-19 mm) to evaluate how the tissue affects the peak intensity and ratio. Thick tissues attenuated both 620 and 700 nm light, with more attenuation at 620 nm than 700 nm. Although this spectral distortion shifted the pH calibration curve, the effect could be corrected for using a scintillator film region with no pH indicator layer as a spectral reference. The sensor was attached to an orthopedic plate affixed to a human cadaveric tibia and imaged through tissue. This approach provides both high spatial resolution from focused X-ray excitation and surface chemical specificity from the indicator dye, providing a tool for imaging local pH through tissue.

X-ray excited luminescent chemical imaging (XELCI) for non-invasive imaging of implant infections.

X-ray excited luminescent chemical imaging (XELCI) uses a combination of X-ray excitation to provide high resolution and optical detection to provide chemical sensing. A key application is to detect and study implant-associated infection. The implant is coated with a layer of X-ray scintillators which generate visible near infrared light when irradiated with an X-ray beam. This light first passes through a pH indicator dye-loaded film placed over the scintillator film in order to modulate the luminescence spectrum according to pH. The light then passes through tissue is collected and the spectral ratio measured to determine pH. A focused X-ray beam irradiates a point in the scintillator film, and a pH image is formed point-by-point by scanning the beam across the sample. The sensor and scanning system are described along with preliminary results showing images in rabbit models.

Implantable strain sensor to monitor fracture healing with standard radiography.

Current orthopaedic clinical methods do not provide an objective measure of fracture healing or weight bearing for lower extremity fractures. The following report describes a novel approach involving in-situ strain sensors to objectively measure fracture healing. The sensor uses a cantilevered indicator pin that responds to plate bending and an internal scale to demonstrate changes in the pin position on plain film radiographs. The long lever arm amplifies pin movement compared to interfragmentary motion, and the scale enables more accurate measurement of position changes. Testing with a human cadaver comminuted metaphyseal tibia fracture specimen demonstrated over 2.25 mm of reproducible sensor displacement on radiographs with as little as 100 N of axial compressive loading. Finite element simulations determined that pin displacement decreases as the fracture callus stiffens and that pin motion is linearly related to the strain in the callus. These results indicate that an implanted strain sensor is an effective tool to help assess bone healing after internal fixation and could provide an objective clinical measure for return to weight bearing.

Tuning Localized Surface Plasmon Resonance Wavelengths of Silver Nanoparticles by Mechanical Deformation.

We describe a simple technique to alter the shape of silver nanoparticles (AgNPs) by rolling a glass tube over them to mechanically compress them. The resulting shape change in turn induces a red-shift in the localized surface plasmon resonance (LSPR) scattering spectrum and exposes new surface area. The flattened particles were characterized by optical and electron microscopy, single nanoparticle scattering spectroscopy, and surface enhanced Raman spectroscopy (SERS). AFM and SEM images show that the AgNPs deform into discs; increasing the applied load from 0 to 100 N increases the AgNP diameter and decreases the height. This deformation caused a dramatic red shift in the nanoparticle scattering spectrum and also generated new surface area to which thiolated molecules could attach as evident from SERS measurements. The simple technique employed here requires no lithographic templates and has potential for rapid, reproducible, inexpensive and scalable tuning of nanoparticle shape, surface area, and resonance while preserving particle volume.