Dental caries is the breakdown of tooth enamel that causes cavities and is the most common chronic disease in children and adolescents between the ages of 6 and 19 years. Therefore, much work is being done to combat caries. We believe that the scanning fiber endoscope (SFE) developed in our lab can be used to improve the detection of early caries in children. The SFE is a small flexible probe that scans low-powered RGB laser light. It performs high-contrast high-resolution imaging and dual laser fluorescence spectroscopy with 405- and 532-nm laser excitation on both in vitro and in vivo teeth. My project focuses on the development of a calibration standard to use with the SFE in an upcoming clinical trial to be conducted at the UW Center for Pediatric Dentistry. A photostable calibration standard was created to match the fluorescence pattern of dental enamel. Spectral measurements of the standard will be taken before and after collecting data from patients' teeth with the SFE to monitor the instrument's behavior. This will allow us to account for drift in measurements and ensure that we can draw accurate conclusions from our data. 

Deadly and leaving patients with little time to live, pancreatic cancer is the third largest cause of deaths due to cancer in the US. Early detection and research of the disease beyond current capabilities are crucial and could be achieved by imaging fully intact pancreatic tissue core biopsies (TCBs) in three-dimensions (3D). Hence, The Human Photonics Laboratory is developing a microfluidic device to process fully intact TCBs (L=0.5-2.0 cm, d=200-1200 µm) in an all-in-one approach. Simultaneously, the device transports the TCB towards a 3D imaging platform for the end goal of enhancing cancer diagnosis. To achieve this goal, each step must be fit and performed in the small device. Typically, microfluidic devices are microfabricated with rectangular cross-sectional channels and used with no larger than cells or cell slurries, but never TCBs. To prepare TCBs for a pathologist in a microfluidic device, transportation is done in curved, circular cross-sectioned channels (CCCs)—both of which have never been achieved before this project. The channels’ cross-sections are circular to produce an intuitive Poiseuille flow profile. TCBs require a smooth directional change due to larger length and diameter of a TCB. Therefore, curved channels are necessary for direction changes and an optimized device footprint. Traditional microfabrication methods are not practical due to the time and precision required for mircofabricatation of a smooth CCC. Therefore, a new simplified method to fabricate microfluidic devices was developed to allow for CCCs and transport availability for TCBs. This new method allows for microfluidic devices containing CCCs to be fabricated in less than one hour and can provide channels that curve in 3D. Transportation of TCBs through CCCs is demonstrated for planar and multi-dimensional direction changes. Minimized channel lengths required to successfully change a TCB’s direction by 90 and 180 degrees were determined to reduce the microfluidic device’s footprint.

Von Willebrand factor (VWF) is a multimeric blood glycoprotein involved in the process blood clotting. Its primary function is to be an adhesive proteins that platelets can bind to a wound site. In the Sniadecki lab, VWF is used as a matrix protein for capturing platelets under shear flow in a microfluidic device for blood coagulation studies. Currently, VWF is purchased from a company and coated on the surface of the microchannels. High molecular weight VWF has a higher ability to form a clot, while low molecular weight VWF has a lower ability. Since the VWF comes from a commercial source, the purity of its molecular weight weight is unknown. The goal of my project is to improve the VWF coating by purifying VWF from Enzyme Research. We hypothesized that low molecular weight VWF does not support clot formation and that high molecular weight VWF will show more clot formation than the control vWF in the microfluidic device. My research efforts have been to purify VWF using the fractionation gel filtration technique developed by Professor Dominic Chung’s lab at the Puget Sound Blood Center. The gel filtration technique can isolate the vWF according to size. Gel filtration was done using a 10x10 Sephacryl 500 Column. After the filtration process, vWF of different molecular weights are then applied primarily to the surface of PDMS so that platelets can bind to them under fast flow, in order to measure forces of platelet clotting. We found that there is a link between the molecular weight of the vWF and the rate of clot formation. Low molecular weight vWF does not support clot formation and that high molecular weight vWF will show more clot formation than the control vWF.

Childhood caries, also known as tooth decay, is on the rise worldwide and has increased the demand for portable technologies that can quickly and accurately detect and diagnose early stage carious lesions. These lesions, if identified at an early stage, can be reversed with remineralization treatments, education, and improvements in home care. A multi-modal optical prototype was previously developed at the University of Washington’s Human Photonics Laboratory for the detection and diagnosis of early occlusal caries demineralization in vivo. The device uses a 405-nm laser as an illumination source to obtain high-resolution images that can better guide the operator to identify a suspicious enamel region. When the suspicious region is located, the device can be switched to perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser excitations generating an auto-fluorescence (AF) ratio with the main goal of diagnosing the start of early caries. Recent research had the device test in vivo on 7 children’s teeth with clinically diagnosed carious, non-cavitated lesions. Using the 405-nm scanned light source, lesion depth was visually estimated by the clinician. Within a month, the clinician assessed the maximum drill depth after the tooth had been drilled and the spectroscopic data acquired. The researcher and clinicians were masked from previous measurements in a blinded study protocol. Using the drill depth as the gold standard, the researcher assessed the preliminary results of the 7 patients to see if the severity or depth of the lesion could be compared quantitatively by using the device. Preliminary results indicate that the quantitative AF spectrum of a tooth correlates with the severity of demineralization as assessed by the clinician after drilling.

Lead zirconate titanate (PZT) is a piezoelectric material commonly used in sensors and actuators. The goal of this project is to formulate a process of producing PZT nanoparticles to be imbedded in a polymer ink that could be used in a 3D printer. The particles must be small enough to make a homogenous colloidal ink that will not obstruct the printer nozzle. My project analyzes the synthesis procedure in detail and how it can affect the piezoelectric properties and morphology of the product. Hydrothermal synthesis was performed at 200 degrees Celsius to control particle size, agglomeration, and chemistry of the PZT particles. It was found that the previous method of synthesis produced particles on the order of 1 to 3 microns in size. In order to reduce particle size below 1 micron I have proposed to reduce time spent growing the particles at elevated temperatures.

Methods and devices used for pressure measurement are extremely varied and typically do not offer the ability to operate accurately when subjected to pressure values outside of the standard operating range. Typical methods and devices used for pressure measurement at low levels of stress, such as those exerted by biological cells, are prohibitively expensive with quite complex manufacturing processes. This research focused on characterizing a device constructed of carbon nanotubes, paper, and polydimethylsiloxane (PDMS), for use as a pressure sensing smart skin with exceptional sensitivity. The geometry and quantum mechanical properties of carbon nanotubes make them highly sensitive to strain. The effects of various factors such as temperature, tension, and environmental air pressure on the electrical resistance of the device were recorded over time to determine its response to its ambient surroundings. Thermal fluctuation, vacuum chamber, mechanical deflection, and control tests were carried out on the sensor to identify its gauge factor and tendency towards hysteresis. The effectiveness of spin- and dip-coating at forming a suitable PDMS layer was investigated. Tests with no PDMS on the sensor were also run. The sensors resistance is directly affected by vacuum pressure and temperature. Increasing the thickness of the PDMS layer reduces the sensitivity to temperature. By characterizing the sensors behavior at various temperatures and pressures, a calibration algorithm is recommended in order to isolate the effect that strain has on the sensors resistance. This easily manufactured, nanostructured sensor for measuring pressure at very low values is an effective tool for research in many fields due to its scalability and ease of use.

Most important biological interactions with the environment occur at interfaces such as a lipid cellular membrane. These interactions are often dynamic and show time-dependent changes in material properties (e.g, viscoelastic properties). A detailed understanding of biomechanical properties at molecular and subcellular level is vital for basic understanding of normal/abnormal biological processes. Indeed, abnormal changes in the mechanics of biological systems are often indicators of pathophysiological states. For example, amyloid beta peptide (Aβ), a protein present in neural plaques formed in Alzheimer’s disease and cholesterol are reported to alter cell membrane mechanical properties leading to neuronal degeneration. Atomic force microscopy (AFM) provides an ideal tool to image the structure and examine the mechanics in physiologically relevant medium. We have created a new cantilevered probe for AFM for the study of nano-to-microscale mechanics of biological interfaces. We used it to examine the role of Aβ1-42 insertion in DPPC lipid monolayers and its effect on the viscoelastic properties that underlie altered membrane fluidity. Real-time analysis of mechanical properties of the model membrane monolayer in the presence of Aβ1-42 shows a decrease in the viscosity of the monolayers over time, consistent with increased membrane fluidity. The finding is consistent with changes in membrane permeability due to Aβ-inserted pores.

We study silica microspheres assembled into a single packed layer, in particular, using acoustic vibrations to assist the assembly process. This single layer of spheres will be used in microsphere-based metamaterial photoacoustic experiments. By adding acoustic waves to the assembly method, it is expected that the microspheres will pack with fewer defects than traditional methods. The wedge-shaped cell self-assembly method is used to form the crystals. Water containing microspheres is left to dry between two angled glass slides. When the liquid solution between the slides evaporates, capillary forces pull the microspheres toward the water line where they will closely pack together. Our research combines this technique with acoustic vibration of the fluid generated from a loudspeaker. An experimental setup was designed with the speaker placed close to the slides, generating white noise at different frequencies and decibel levels. This sound will theoretically shake the spheres as the water evaporates and improve particle ordering. Understanding the dynamics of this material could potentially lead to their use in items such as signal processing devices, protective coatings and armors, and biosensors.

With the increasing application of electromyography (EMG) sensor systems (method to measure electric potential generated during muscle contraction) in automation and robotics control, investigations on an effective user-interface are essential. My goal is to investigate solutions that enable valid communications between users and a system under complex sensor behaviors. If a user is unable to complete a motion or to continuously generate the proper control signal, the interface detects the desired signal and reproduces the intended result. There are two components in this research: (i) finding the behaviors of EMG signal, which determines the way users interact with the system, and (ii) adjusting or approximating the output to an ideal signal through visual feedback. To address the first task, I have processed the EMG sensor signal by implementing an analog and digital filter system to achieve a reliable EMG signal. Towards this, I created a LabView program to generate a signal of a desired function (e.g. Asin(ωt+φ)), by detecting the magnitude, frequency, and phase information of the input signal. Currently, my signal generation program works with a sinusoidal waveform in the presence of noise. However, physical EMG signals are not perfect sinusoidal waves. Using my program, the interface between the human-generated EMG signal and the computer generated result can be explored, with the goal of using EMG signals to control the computer generated signals. This will be accomplished by modifying the detection algorithm developed for the sinusoidal inputs.

Specialty polymer optical fiber requires incredibly tight diameter control of ~ 1 um to minimize signal attenuation. Temperature profiles within cylindrical furnaces used to bring about necking of preforms to be drawn into fiber cause the formation of convective oscillating cells. The resulting variations in heat flux into the preform likewise cause oscillations in the fiber exit diameter. For preforms of sufficient diameter, matching a prescribed temperature profile within the necking region of the furnace leads to fiber diameter oscillations of predictable amplitude and frequency. The necessary temperature profile and the extent to which this affect can be achieved varies with the geometry of the inside of the furnace; namely, preform shape and diameter. To maintain ease of manufacturability, accommodating various preform diameters is necessary. By drawing fiber under temperature profiles described above, it is hoped that coupling these well-understood and reproducible oscillations with a PID feedback controller using draw speed to regulate exit diameter and tuned to these specific oscillations, we will attain diameter control within 1 um for the ideal case of a large 1” diameter preform. Preforms of varying diameters will be drawn under ideal temperature profiles, with the fiber diameter recorded. When steady oscillations are achieved, a PID controller tuned to their amplitude and frequency will be turned on, and the diameter response will be observed. Earlier work indicated that the process is highly sensitive to temperature and draw speed, and previous attempts to achieve 1 um diameter control using only PID feedback or temperature profile regulation proved inadequate. This work will determine if a combined approach advantageous, and provide insight into the more challenging control problems associated with specialty fiber preforms of smaller diameter. If achievable, this would allow inexpensive production of advanced graded-index fiber.

Microfluidic devices containing microposts, which are used to measure platelet contractile force, are fabricated using a flexible polymer called polydimethylsiloxane (PDMS). PDMS possesses many advantageous characteristics including being non-toxic, optically clear, and typically desirable mechanical properties. However, PDMS is limited as a microfluidic device given its hydrophobicity, or strong ability to repel water. This characteristic is especially problematic when PDMS is used in microfluidic devices intended to analyze blood samples. Blood is protein rich, so the hydrophobicity in PDMS leads to significant biofouling, or biological accumulation, on the surface. Moreover, the range of mechanical properties available with PDMS is limited. This limits the utility of microposts as a platelet force sensing tool since the microposts may be either too stiff or too soft to adequately measure contractile force. The work presented herein sought to circumvent these challenges by replacing PDMS with polyurethane (PU). PU was chosen because its mechanical properties can be modified to adapt to a range of platelet contractile forces from, for example, hyper- and hyporeactive platelets. First, a range of PU types was explored. From these, a commercial grade PU entitled ClearFlex was found to be the best. As a result, it was used in subsequent characterizations, and an optimized protocol for casting microfluidic devices from ClearFlex was developed. The next steps are to test the mechanical, optical, and biological properties of ClearFlex compared to PDMS. The final PU microfluidic device will be developed into a diagnostic tool specifically for clotting disorders.

The incidence of esophageal adenocarcinoma (EAC) is increasing world-wide. Early and specific detection of EAC and its precursor non-cancerous stages, particularly Barrett’s esophagus (BE), is crucial to treatment and long-term survival. Currently, endoscopy coupled with serial biopsy is the gold standard for diagnosis. Standard endoscopy lacks specificity. BE presents as a salmon-reddish discoloration against the pale-pink color of healthy esophageal tissue. Cardia intestinal metaplasia (CIM) arising from gastric tissue is visually similar; both develop in response to prolonged acid reflux and are often confused during histological analysis. However, CIM is unrelated or even negatively correlated to EAC development. Distinguishing between BE and CIM at the gastroesophageal junction is crucial to minimizing the use of invasive biopsy. Targeting molecular markers specific to disease state can enhance specificity. Fluorescent dyes attached to targeting molecules are used to label these biomarkers. Instrumentation designed for in-vivo fluorescent imaging, like the ultrathin, flexible, multimodal scanning fiber endoscope (SFE) can then excite and image the fluorescent dyes, effectively red-flagging abnormal regions for both enhanced biopsy capture and point-of-care diagnosis. Reliance upon one biomarker alone is ineffective due to patient heterogeneity, as expression varies between patients due to factors including genetic background and lifestyle. Multiple biomarkers are necessary for specificity. The purpose of our work is to first demonstrate that the three cell surface biomarkers, EGFR, C-MET, and ERBB2, well established in other cancers, are appropriate for early EAC or BE diagnosis. We plan to demonstrate the simultaneous application of all three biomarkers to provide added specificity and sensitivity for diagnosis and ultimately prognosis. These goals will be achieved through immunofluorescent staining of cell lines and paraffinized tissue samples excised from patients. The efficacy of using multiple markers will be assessed through image processing and statistical analysis.

A major application of micropost technology is the measurement cellular traction forces to quickly diagnose potential medical issues. Currently, polydimethylsiloxane (PDMS) is the material used to fabricate micropost arrays using an intensive three-day double casting procedure. The process begins with casting a negative PDMS mold from a very delicate silicon master array. Negative molds cast from this master require surface treatment overnight via silanization in a vacuum, and then final casting requires an additional overnight cure. The silanization step cannot be forgone in the current process; doing so would result in a micropost array that is inseparable from its negative mold. Additionally, the double casting process must be repeated for every new array in each experiment since PDMS negative molds are not reusable due to degradation by repeated surface treatments. In order to improve upon the micropost fabrication process and widen the scope of applications beyond academia, my colleagues and I are developing a technique to cast PDMS micropost arrays directly from a reusable polyurethane negative mold. Our primary goal is to determine the reusability of polyurethane negative molds in casting PDMS micropost arrays without repeated silanization. To characterize reusability, I will use scanning electron microscopy to measure and quantify the dimensions of the microposts in the arrays in comparison to the silicon master. This step will be repeated for each array reproduced from the polyurethane negative mold as a quantitative measure of its reusability. The creation of a reusable polyurethane negative mold would completely eliminate the intermediate PDMS negative mold and subsequent silanization requirements. Therefore, this replacement would reduce the time required to create a PDMS micropost array from three days to eight hours. Moreover, a polyurethane negative mold could also allow for the simultaneous fabrication of multiple arrays, thus further reducing the time requirement for fabrication significantly.

Polydimethylsiloxane (PDMS) micro and nanoposts have been widely used to measure traction forces in cells, however, the mechanical properties of PDMS micro and nanoposts remain unclear. The current measurements of our lab’s PDMS posts are calculated based on the bulk or global properties of PDMS versus the local modulus, which matters at the cellular scale. It is important to denote this difference because cells actively sense their local physical microenvironments, not the global environment or bulk modulus. In this project, I fabricated specified polydimethylsiloxane micro and nanoposts, which are used in lab to measure cellular forces, and examined the mechanical properties (spring constant and Young’s modulus) of these micro and nanoposts, taking into account the fact that cells only sense their local microenvironments which means experimentally finding the properties of PDMS at the micro and nanoscale. To fully characterize this hydrogel, I utilized atomic force microscopy (AFM) to provide a local constant force (0-60nN for calibration), scanning electron microscopy (SEM) to calculate the dimensions of specified posts of the micro and nanoposts at 110⁰ C at different curing times. Future research utilizing PDMS micro and nanoposts to measure cell forces will take into account these correction values for exact force measurements. These findings provide an update and more thorough way to calculate the mechanical properties of the PDMS micro and nanoposts used in lab.