LITE-1 mediates behavioral responses to X-rays in Caenorhabditis elegans.

Rapid sensory detection of X-ray stimulation has been documented across a wide variety of species, but few studies have explored the underlying molecular mechanisms. Here we report the discovery of an acute behavioral avoidance response in wild type Caenorhabditis elegans to X-ray stimulation. The endogenous C. elegans UV-photoreceptor protein LITE-1 was found to mediate the locomotory avoidance response. Transgenic expression of LITE-1 in C. elegans muscle cells resulted in paralysis and egg ejection responses to X-ray stimulation, demonstrating that ectopic expression of LITE-1 can confer X-ray sensitivity to otherwise X-ray insensitive cells. This work represents the first demonstration of rapid X-ray based genetically targeted (X-genetic) manipulation of cellular electrical activity in intact behaving animals. Our findings suggest that LITE-1 has strong potential for use in this minimally invasive form of neuromodulation to transduce transcranial X-ray signals for precise manipulation of neural activity in mammals, bypassing the need for invasive surgical implants to deliver stimulation.

Contrast agents for x-ray luminescence computed tomography.

Imaging probes are an important consideration for any type of contrast agent-based imaging method. X-ray luminescence imaging (XLI) and x-ray luminescence computed tomography (XLCT) are both contrast agent-based imaging methods that employ x-ray excitable scintillating imaging probes that emit light to be measured for optical imaging. In this work, we compared the performance of several select imaging probes, both commercial and self-synthesized, for application in XLI/XLCT imaging. Commercially available cadmium telluride quantum dots (CdTe QDs) and europium-doped gadolinium oxysulfide (GOS:Eu) microphosphor as well as synthesized NaGdF4 nanophosphors doped with either europium or terbium were compared through their x-ray luminescence emission spectra, luminescence intensity, and also by performing XLCT scans using phantoms embedded with each of the imaging probes. Each imaging probe displayed a unique emission spectrum that was ideal for deep-tissue optical imaging. In terms of luminescence intensity, due to the large particle size, GOS:Eu had the brightest emission, followed by NaGdF4:Tb, NaGdF4:Eu, and finally the CdTe QDs. Lastly, XLCT scans showed that each imaging probe could be reconstructed with good shape and location accuracy.

Focused x-ray luminescence imaging system for small animals based on a rotary gantry.

SIGNIFICANCE: The ability to detect and localize specific molecules through tissue is important for elucidating the molecular basis of disease and treatment. Unfortunately, most current molecular imaging tools in tissue either lack high spatial resolution (e.g., diffuse optical fluorescence tomography or positron emission tomography) or lack molecular sensitivity (e.g., micro-computed tomography, µCT). X-ray luminescence imaging emerged about 10 years ago to address this issue by combining the molecular sensitivity of optical probes with the high spatial resolution of x-ray imaging through tissue. In particular, x-ray luminescence computed tomography (XLCT) has been demonstrated as a powerful technique for the high-resolution imaging of deeply embedded contrast agents in three dimensions (3D) for small-animal imaging. AIM: To facilitate the translation of XLCT for small-animal imaging, we have designed and built a small-animal dedicated focused x-ray luminescence tomography (FXLT) scanner with a µCT scanner, synthesized bright and biocompatible nanophosphors as contrast agents, and have developed a deep-learning-based reconstruction algorithm. APPROACH: The proposed FXLT imaging system was designed using computer-aided design software and built according to specifications. NaGdF4 nanophosphors doped with europium or terbium were synthesized with a silica shell for increased biocompatibility and functionalized with biotin. A deep-learning-based XLCT image reconstruction was also developed based on the residual neural network as a data synthesis method of projection views from few-view data to enhance the reconstructed image quality. RESULTS: We have built the FXLT scanner for small-animal imaging based on a rotational gantry. With all major imaging components mounted, the motor controlling the gantry can be used to rotate the system with a high accuracy. The synthesized nanophosphors displayed distinct x-ray luminescence emission, which enables multi-color imaging, and has successfully been bound to streptavidin-coated substrates. Lastly, numerical simulations using the proposed deep-learning-based reconstruction algorithm has demonstrated a clear enhancement in the reconstructed image quality. CONCLUSIONS: The designed FXLT scanner, synthesized nanophosphors, and deep-learning-based reconstruction algorithm show great potential for the high-resolution molecular imaging of small animals.

X-ray excited luminescence spectroscopy and imaging with NaGdF4:Eu and Tb.

X-ray excited optical luminescence from nanophosphors can be used to selectively generate light in tissue for imaging and stimulating light-responsive materials and cells. Herein, we synthesized X-ray scintillating NaGdF4:Eu and Tb nanophosphors via co-precipitate and hydrothermal methods, encapsulated with silica, functionalized with biotin, and characterized by X-ray excited optical luminescence spectroscopy and imaging. The nanophosphors synthesized by co-precipitate method were ∼90 and ∼106 nm in diameter, respectively, with hydrothermally synthesized particles showing the highest luminescence intensity. More importantly, we investigated the effect of thermal annealing/calcination on the X-ray excited luminescence spectra and intensity. At above 1000 °C, the luminescence intensity increased, but particles fused together. Coating with a 15 nm thick silica shell prevented particle fusion and enabled silane-based chemical functionalization, although luminescence decreased largely due to the increased mass of non-luminescent material. We observed an increase in luminesce intensity with temperature until at 400 °C. At above 600 °C, NaGdF4:Eu@SiO2 converts to NaGd9Si6O26:Eu, an X-ray scintillator brighter than annealed NPs at 400 °C and dimmer than NPs synthesized using the hydrothermal method. The particles generate light through tissue and can be selectively excited using a focused X-ray source for imaging and light generation applications. The particles also act as MRI contrast agents for multi-modal localization.

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.

In situ preparation of gold-polyester nanoparticles for biomedical imaging.

The synthesis and application of gold nanoparticles (AuNPs) have attracted much attention due to their interesting optical and chemical properties, as well as their utility in imaging, therapeutics, sensors, electronics, and catalysis. AuNPs are synthesized using multiple approaches, followed by chemical modification or encapsulation, to enhance their colloidal stability, biocompatibility, and targeting. Here, we report the one-step synthesis of gold-polyester nanoparticles for use as an imaging agent. The AuNPs were prepared inside polymeric NPs by means of ultraviolet irradiation of a gold salt in the presence of Irgacure I-2959 photoinitiator. We monitored the kinetic growth and nucleation of AuNPs (in vitro and ex vivo) over time using spectral analysis. Moreover, we investigated the cytotoxicity, localized plasmonic surface resonance (LSPR), and cellular imaging capabilities of the Au-polyester nanoparticles. The resulting Au-polyester NPs were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to probe their chemical structure, size, zeta potential (ζ), and morphology, respectively. Furthermore, in vitro experiments showed that the NP formulation is stable over time and exhibits negligible toxicity against 3T3 fibroblast and U-87 MG glioblastoma cells. The results also demonstrated that the Au-polyester NPs exhibit excellent cellular imaging properties. This one-step strategy goes beyond current syntheses of gold-polyester nanoparticles because it can be used to synthesize the imaging agent in situ (i.e., in living cells) in lieu of conventional ex situ approaches.

X-ray luminescence imaging for small animals.

X-ray luminescence imaging emerged for about a decade and combines both the high spatial resolution of x-ray imaging with the high measurement sensitivity of optical imaging, which could result in a great molecular imaging tool for small animals. So far, there are two types of x-ray luminescence computed tomography (XLCT) imaging. One uses a pencil beam x-ray for high spatial resolution at a cost of longer measurement time. The other uses cone beam x-ray to cover the whole mouse to obtain XLCT images at a very short time but with a compromised spatial resolution. Here we review these two methods in this paper and highlight the synthesized nanophosphors by different research groups. We are building a focused x-ray luminescence tomography (FXLT) imaging system, developing a machine-learning based FXLT reconstruction algorithm, and synthesizing nanophosphors with different emission wavelengths. In this paper, we will report our current progress from these three aspects. Briefly, we mount all main components, including the focused x-ray tube, the fiber detector, and the x-ray tube and x-ray detector for a microCT system, on a rotary which is a heavy-duty ring track. A microCT scan will be performed before FXLT scan. For a FXLT scan, we will have four PMTs to measure four fiber detectors at two different wavelengths simultaneously for each linear scan position. We expect the spatial resolution of the FXLT imaging will be around 100 micrometers and a limit of detection of approximately 2 µg/mL (for Gd2O2S:Eu).