Ultrasonography has been widely applied in imaging tissues and organs for diagnosing diseases due to its capability of serving as a real-time, non-invasive, portable, and radiation-free tool. Ultrasound imaging has demonstrated significant value in diagnosing and monitoring periodontitis that can improve the limitations of conventional methods such as radiography and periodontal probing. While radiography provides excellent sensitivity to hard tissues, it uses ionizing radiation and is limited in stratifying disease in soft tissues. Periodontal probing is time-consuming and painful for patients, and inter-operator variation in probing can exceed 40%.In this dissertation, the first part reviews the materials and mechanisms of ultrasound transducers. The second part includes our work that developed a wearable skin-conformal ultrasonic phased array patch for continuous haemodynamics monitoring in deep tissue. The penetration depth is up to 14 cm beneath the skin, allowing accessing biological signals from central organs. The third part includes our work that developed a toothbrush-shaped ultrasound transducer to image all 28 human teeth with its miniaturized size and side-facing design. The results showed that the full-mouth imaging process by the miniaturized transducer is reproducible in a small cohort of five subjects. Besides imaging anatomical structures, blood flow of the supraperiosteal artery in human gingiva was measured by color and spectral Doppler to provide hemodynamic information. The fourth part includes our work that mapped the location and physiology of the greater palatine artery (GPA) in a 3D manner. Mapping the trajectory of the GPA in the hard/soft palate is clinically important to evaluate the available palate donor tissue during soft tissue autografting and to avoid sectioning of the GPA during the surgery. The fifth part summarizes our work of reconstructing sparse array ultrasound imaging with deep learning algorithms. Sparse array ultrasound imaging has presented potential in reducing the fabrication cost, energy consumption and electrical system complexity. However, sparse array imaging suffers from inevitable artifacts due to grating lobes. We developed a convolutional neural network (CNN)-assisted sparse array using only 16 channels out of a 128-channel transducer, in which the pitch is eight times that of the dense array.

Periodontitis is a public issue and imaging periodontal pocket is important to evaluate periodontitis. Dental ultrasound imaging has been increasingly studied for the diagnosis of periodontitis. Previous studies showed that the distances between several anatomical landmarks including alveolar bone crest (ABC), cementoenamel junction (CEJ), and gingival margin (GM) are significantly different among the healthy and diseased groups. However, these works were limited to anterior teeth (incisors and canines) due to the bulky size of the transducer. Moreover, ultrasound imaing lack the contrast to resolve the periodontal probing depth (PPD) due to the lack of contrast. In this dissertation, I will focus on developing compact photoacoustic and ultrasound imaging techniques that aims at full-mouth periodontal imaging. In Chapter 1, I will demonstrate a simple, yet versatile method named “Scissors” to help synchronize ultrasound open platforms (UOPs) for photoacoustic imaging with improved imaging quality. Scissors is a programmed function that can cut or add a fixed delay to RF data and thus synchronize it before reconstruction for real time imaging. In Chapter 2, I will report a new method named interleave-sampled PA imaging that enables high-frequency imaging with a relatively low sampling rate, e.g., a 41.67-MHz sampling rate with a 30-MHz transducer. High-frequency photoacoustic (PA) imaging (>20 MHz) requires data acquisition (DAQ) with commensurately high sampling rate, which leads to hardware challenges and increased costs. This method harnesses two acquisitions at a low sampling rate to effectively double the sampling rate for high-frequency imaging. It modulates the delay of the light pulses and can thus be applied to any PA DAQ system. In Chapter 3, I will report a clinical “hockey-stick”-style transducer integrated with fibers for periodontal photoacoustic imaging, which allows us to measure the periodontal probing depth (PPD). The unique angle shape of hockey-stick transducer allows it to image more posterior teeth than regular linear transducers. We used cuttlefish ink to label the periodontal pocket as a photoacoustic contrast agent. We characterized the imaging system, and then measured the pocket depth of 35 swine teeth. Three raters evaluated the performance of the hockey-stick transducer. The measurements between the Williams probing (gold standard) and the photoacoustic methods were blinded but highly correlated. We showed a bias of ~0.3 mm for the imaging-based technique versus Williams probing. The minimum inter-reliability was over 0.60 for three different raters of varying experience suggesting that this approach to measure periodontal pocket is reproducible. Finally, we imaged the three pre-molars of a human subject. We could access additional upper and posterior teeth than conventional linear transducers. In chapter 4, we characterized a transducer that can image the entire human mouth including assessment of periodontal pockets via a combination of photoacoustic and ultrasound imaging. Unlike conventional transducer design, this device has a toothbrush-shaped form factor with a side-view transducer to image molars (total size: 1 x 1.9 cm). A laser diode was integrated as the light source to reduce the cost and size and facilitates clinical transition. The in vivo imaging of a molar of a periodontal patient demonstrated that the transducer could image in the posterior area of gum in vivo; the value determined by imaging was within 7% of the value measured clinically. In Chapter 5, we used a commercially available 9-MHz hockey-stick transducer for clinical periodontal imaging. The hypothesis is that the distances between several anatomical landmarks including alveolar bone crest (ABC), cementoenamel junction (CEJ), and gingival margin (GM) are significantly different among the healthy and diseased groups. Here, the unique angle design of the hockey-stick transducer allows it to image posterior teeth of patients. We imaged 13 subjects including 53 premolars, 30 molars, and 79 incisors and canines. The inter-examiner tests demonstrated that the clinical parameters obtained from ultrasound imaging with the hockey-stick transducer were reproducible. We further compared the measurements from ultrasound imaging and periodontal probing among periodontally healthy and diseased patients. Notably, the average imaging-based gingival recession (iGR) measurements were -1.12 mm (i.e., 1.12 mm above the CEJ) for gingivitis and Stage I periodontitis, and -0.56 mm for Stage III patients, demonstrating a significant increase of gingival recession in patients with severe periodontitis (Student t-test, unpaired, two-tailed, p < 0.0001). Our results showed that ultrasound imaging has value in periodontal diagnosis.

Cardiovascular diseases are a leading cause of death. Although the existing therapies can lower early mortality rates and reduce the risk of further heart attacks, patients can suffer from existing damage for their rest of life due to the very limited regeneration capacity of the heart. Therefore, many efforts have been made to regenerate and repair cardiac tissue via stem cells. However, stem cell therapy in cardiovascular diseases treatment is challenged by mis-injection, poor survival, and low retention of cells. Solutions to these challenges can be multimodal imaging contrast agents, sustained or controlled drug delivery, and magnetic manipulation of cells, all of which bring attention to nanotechnology. In this dissertation, I will demonstrate how nanotechnology can benefit stem cell therapy with inorganic silicon-based nanomaterials and mesenchymal stem cells. Mesenchymal stem cells (MSCs) are used in this dissertation because they are particularly promising due to their abundance, potent proliferation, and multi-lineage differentiation capacity.

First, the role of hybrid nanoparticles in multi-modal imaging and theranostic nanomedicine and an introduction to medical imaging modalities and therapeutic methods are reviewed.

The second chapter describes a novel discoid silica nanoparticle, which offered the most enhanced ultrasound signal of MSCs compared to three other classical silica structures including Stober silica, mesoporous silica, and mesocellular foam silica.

The third chapter first introduces the biocompatibility of silicon carbide nanomaterials. Silicon carbide nanowires inhibited proliferation and differentiation of MSCs while silicon carbide nanoparticles showed no impact on the MSCs’ viability and functions. In the second part, the potential of silicon carbide nanoparticles in stem cell long-term imaging/tracking via photoacoustic and photoluminescence imaging is shown.

The fourth chapter studies the drug adsorption behavior of organosilica nanoparticles with intrinsic amine groups.

Last, a biocompatible multi-functional silica-iron oxide nanoparticle is designed. This nanoparticle increases MRI and ultrasound contrast, cell viability, and retention of MSCs. In vivo studies show that labeling stem cells with this nanoparticle increase the stem cell therapy efficacy in murine animals injured by a ligation/reperfusion surgery.

Advancements in nanomedicines have enabled breakthroughs in both disease diagnosis and treatment, including ultrasound bioimaging and cancer immunotherapy. On the one hand, nanomedicines of various formulations have been used as ultrasound contrast and greatly improved the ultrasound imaging quality. However, an ideal ultrasound contrast agent— that has tunable, modifiable surface, biodegradability, biocompatibility, and sufficient stability (half-life) for transient ultrasound imaging— is yet to be explored. On the other hand, to overcome the limitations of immune checkpoint blockage therapy,— immune-related toxicity and inefficacy in immune-suppressive tumors— nanomedicines have been rationally designed to trigger immunogenic cell death (ICD) of tumor cells. However, further work is necessary to develop ICD-inducing nanomedicines that are biodegradable, biocompatible, and intrinsically potent in inducing ICD. In this dissertation, I will discuss how creating novel nanomedicines can benefit both ultrasound imaging and cancer immunotherapy.First chapter reviews applications of nanomedicines in both ultrasound imaging and ICD-based cancer immunotherapy, with a highlight of successes and limitations of existing nanomedicines. Second chapter describes how sol-gel reaction combined with electrospray can generate biodegradable calcium phosphate micro/nano particles. Effects of synthetic parameters on the particle size and morphology are discussed in detail. The biodegradability of these particles was validated. Third chapter illustrates a bio-inspired, facile, mild, and solution-based synthetic method for creating calcium phosphate micro/nano particles. Systematic investigations on synthetic parameters led to calcium phosphate particles with versatile sizes (396±128 nm to 63±8 µm) and morphologies (hexagonal micro-disc, micro-flower, micro-leaf, nano-butterfly, and nano-ribbon). A “phosphate sponge” mechanism was found to be the key for regulating the particle sizes and morphologies. These calcium phosphate micro/nano particles are proven to be biodegradable, non-toxic, and efficient in creating transient ultrasound contrast. Fourth chapter studies the building of proton sponge nano-assembly (PSNA) optimized for imaging and triggering ICD of cancer cells. By carefully tuning the hydrophilic and hydrophobic components, the self-assembly tendency of PSNA was optimized. In turn, the PSNA with the highest fluorescence, positive surface charge density, intracellular fluorescence, and cancer cell cytotoxicity was achieved. The lysosome rupturing-regulated pyroptosis and necroptosis were triggered by PSNA, suggesting the great potential of the PSNA for anticancer immunity.

Plasmonic nanoparticles (PNs) have unique optical properties that make them particularly attractive for biosensing applications. These properties arise due the localized surface plasmon resonance (LSPR) on their surface which occurs upon light excitation that is typically in the visible region of the electromagnetic spectrum. The absorbing and scattering properties of PNs are derived from the LSPR and are tunable based on the nanoparticle shape, size, and local dielectric environment. Furthermore, the LSPR on PNs enables fluorophores in close proximity (<10 nm) to be quenched through a nanosurface energy transfer (NSET) mechanism. In this dissertation, PNs are utilized to construct three novel biosensing modalities: The first by modulating the shape of PNs in response to enzyme inhibitors; the second by controlling the aggregation state of PNs in response to enzymes that are upregulated in cancers; and the third by turning “on” and “off” the fluorescence of dyes ligated to PNs in response to cell-death signaling enzymes. Specifically, in chapter one the optical properties of plasmonic gold nanorods (AuNRs) are explored in the context of their aspect ratio (length/width) to construct a colorimetric sensor. The LSPR of the AuNRs was highly sensitive to changes in aspect ratio and resulted in a visible color change in solution when incubated with a trypsin inhibitor. The second chapter shows how gold PNs with a thin layer of silver change color in different aggregation states. The aggregation state was determined by how much the PN crosslinker, a cysteine containing peptide/substrate, was digested by trypsin. In chapter three, the surface chemistry of gold nanoparticles is engineered to present self-assembling, coiled-coil peptides. One of the coiled-coil peptides in the pair on the PN surface was conjugated with a fluorophore and turns “on” once released upon digestion by the enzyme caspase-3.

Phenolic materials are naturally occurring molecules present in a wide range of organisms, from fungi to bacterium, and from plants to animals. Their unique structural and chemical features can form covalent or non-covalent interactions with diverse materials including inorganic (e.g., metal ion, metal oxide, silica, gold, silver), organic (e.g., small molecule, polymers), and biomaterials (e.g., proteins, peptides). As phenolic-based material can establish multiple physicochemical interactions such as metal coordination, hydrogen bonding, interactions, hydrophobic interactions, electrostatic interactions, and covalent bonding, diverse phenolic-enabled nanostructures have been developed for biomedical applications such as phenolic nanoparticles, phenolic surface coating, and secondary growth on phenolic surface. In this dissertation, I will explain how phenolic materials can benefit nanomaterials and their role in bioimaging and biosensing applications. First chapter reviews the hybrid gold nanorod-polydopamine nanoparticles which enhance photoacoustic performance more than bare gold nanorod both in in vitro and in vivo. The role of polydopamine shell in protection for gold nanorod is largely described. Second chapter describes the impact of nanoparticle size in photoacoustic and photothermal therapy. Two different-sized gold nanorods were synthesized and coated with polydopamine. These two different sizes of polydopamine-coated gold nanorod show same extinction peak (i.e., 1064 nm); however, the small size of gold nanorod exhibits remarkable photoacoustic and photothermal performance compared to large sized particles. Third chapter studies the polydopamine nanocapsule coated with organic dyes to detect heparin in whole human blood and plasma. The dye-coated polydopamine nanocapsule showed increased photoacoustic signal as a function of heparin concentration due to particle aggregation. In this chapter, tannic acid-mediated di thiol nanoparticles were also introduced to synthesize polydopamine nanocapsules. Fourth chapter investigates the impact of skin tone on biomedical optics. Darker skin tones (i.e., phototype) absorb and scatter more incident light before it reaches to target of interest. To understand the impact of skin tone, we mimic human skin using gelatin-based hydrogel and polydopamine nanoparticles (referred as synthetic melanin) in which they have similar chemical and optical properties compared to real melanin. Using 3D-bioprinted skin phantoms, multiple biomedical optics (photoacoustic, fluorescence, and photothermal) were extensively examined. Fifth chapter includes peptide-driven particle dissociation of gold nanoparticle aggregates for protease detection. Gold nanoparticle aggregation is often hindered by protein corona formation in biofluid, and undesired aggregation can produce false positives. In this chapter, diverse peptide structures were investigated for a matrix-insensitive dissociation platform.

Medical imaging requires the perturbation of biological tissues with external radiation. Clinically, acoustic and optical methods (e.g., ultrasonography, fluorescence imaging) are attractive because they are non-ionizing and offer real-time acquisition with high resolution. Photoacoustic imaging is an emerging hybrid modality that combines the contrast of optics with the penetration of ultrasound. This dissertation presents novel strategies for the detection of aberrant biomarkers through the application of high-frequency ultrasonography, optical methods (i.e., visible/near-infrared absorbance, fluorescence, scattering) and photoacoustic imaging, with primary focuses in periodontal disease and protease detection. First, the preclinical field of photoacoustic imaging is reviewed across various disease areas for image-guided therapy, surgery, and drug delivery. The second chapter describes research advances in photoacoustic imaging specifically for drug delivery. The third chapter reviews progress to date in photoacoustic imaging for dental and oral health applications. In the fourth chapter, the clinical value of high-resolution ultrasonography is demonstrated for noninvasive and chairside measurement of anatomic periodontal metrics in a pilot case control study. Next, this work is extended to imaging the periodontal pocket in a healthy human case study using photoacoustic imaging with a food-grade contrast agent. Chapter six describes the application of a modality-independent algorithmic method for reducing motion artifacts that commonly arise during 3D photoacoustic/ultrasound imaging of the oral environment. Chapter seven describes the chemical development of a modular and activatable contrast agent that produces photoacoustic and fluorescent signal upon proteolytic cleavage by leveraging the intramolecular coupling of dye-peptide conjugates. This work is validated with trypsin as a model protease and subcutaneous photoacoustic imaging of the probe in mice. In chapter eight, the design is honed for targeting proteolytic gingipains secreted by the periodontal pathogen, Porphyromonas gingivalis, with demonstrated photoacoustic imaging of the gingipain-activated probe in ex vivo swine jaws and fluorescent activation in gingival crevicular fluid samples collected from human subjects with periodontal disease. In chapter nine, the dissertation concludes with a foray into the characterization of protein aggregates associated with Alzheimer’s disease. It is demonstrated that multispectral nanoparticle tracking analysis can utilize light scattering at multiple wavelengths to measure the nanoscale distribution of amyloid-β aggregates (size and concentration) in vitro, without interfering dyes and in real time.

Photoacoustic imaging is an emerging modality which the combination of ultrasound and optical imaging. The combination of these two techniques has many advantages including no use of ionizing radiation compared to radiography, high-resolution deep tissue imaging versus optical coherence tomography (OCT), and higher contrast and faster scanning compared to MRI. Most current equipment uses sophisticated and complicated OPO lasers with tuning and stability features inconsistent with broad clinical deployment. Low fluence illumination sources can facilitate clinical transition of photoacoustic imaging because they are rugged, portable, affordable, and safe. In this dissertation, I will present characterization of the commercial available light emitting diode (LED) based photoacoustic imaging in terms of system specifications, light source characterizations, photoacoustic spatial/temporal resolution, and penetration. Since low fluence light source based photoacoustic imaging devices generate low image quality, I will propose a denoising method using a multi-level wavelet-convolutional neural network to map low fluence illumination source images to its corresponding high fluence excitation map. This part of dissertation will show qualitative and quantitative improvements up to 2.20, 2.25, and 4.3-fold for peak signal-to-noise ratio (PSNR), similarity structural index measurement (SSIM), and contrast-to-noise ratio (CNR) metrics. Next, after improving and enhancing the low fluence light source photoacoustic imaging systems, we report molecular and functional imaging application for LED-based photoacoustic imaging . We demonstrate detection of reactive oxygen and nitrogen species (RONS) with a near-infrared (NIR) absorbing small molecule (CyBA) and LED-based photoacoustic imaging equipment. CyBA produces increasing photoacoustic signal in response to peroxynitrite (ONOO−) and hydrogen peroxide (H2O2) with photoacoustic signal increases of 3.54 and 4.23-fold at 50 μM of RONS at 700 nm, respectively. We also introduced photoacoustic imaging as a non-invasive method for detecting early tissue damage that cannot be visually observed while also staging the disease using quantitative image analysis. Finally, Here we introduce polyacrylamide (PAA) hydrogel as a candidate material for fabricating stable phantoms with well-characterized optical and acoustic properties that are biologically relevant over a broad range of system design parameters. These phantoms may also facilitate future standardization of performance test methodology.