Cost-Effective Full-Color 3D Dental Imaging Based on Close-Range Photogrammetry.

Dental imaging plays a crucial role in clinical dental practice. Conventional 2D dental imaging serves general-purpose tasks, such as patient documentation, while high-precision 3D dental scanning is tailored for specialized procedures, such as orthodontics and implant surgeries. In this study, we aimed to develop a cost-effective 3D imaging technique that could bridge the gap between conventional dental photography and high-precision 3D dental scanning, with the goal of improving patient dental care. We developed a 3D imaging technique based on close-range photogrammetry and termed it close-range photogrammetry-based dental imaging (CPDI). We evaluated this technique on both in vitro dental models and in vivo teeth. For dental models, we conducted a parametric study to examine the effects of the depth of field and specular reflection on reconstruction quality. We showed that the optimal results were achieved with an f/5.6 lens and without a circular polarizer for reflection suppression. This configuration generated 3D scans with 57.7 ± 3.2% and 82.4 ± 2.7% of reconstructed points falling within ±0.1 mm and ±0.2 mm error margins, respectively. With such accuracy, these 3D dental models can faithfully represent dental morphology and features. During in vivo imaging, we were able to reconstruct high-quality 3D models of the anterior arch, further demonstrating its clinical relevance. The reconstructed models carry both 3D shapes and detail full-color surface textures, which positions CPDI as a versatile imaging tool in different areas of clinical dental care.

Differentiating methicillin resistant and susceptible Staphylococcus aureus from ocular infections using photoacoustic labeling.

Introduction: Antibiotic resistance in bacterial species constitutes a growing problem in the clinical management of infections. Not only does it limit therapeutic options, but application of ineffective antibiotics allows resistant species to progress prior to prescribing more effective treatment to patients. Methicillin resistance in Staphylococcus aureus is a major problem in clinical infections as it is the most common hospital acquired infection. Methods: We developed a photoacoustic flow cytometer using engineered bacteriophage as probes for rapid determination of methicillin resistance in Staphylococcus aureus with thirteen clinical samples obtained from keratitis patients. This method irradiates cells under flow with 532 nm laser light and selectively generates acoustic waves in labeled bacterial cells, thus enabling detection and enumeration of them. Staphylococcus aureus isolates were classified from culture isolation as either methicillin resistant or susceptible using cefoxitin disk diffusion testing. The photoacoustic method enumerates bacterial cells before and after treatment with antibiotics. Decreasing counts of bacteria after treatment indicate susceptible strains. We quantified the bacterial cells in the treated and untreated samples. Results: Using k-means clustering on the data, we achieved 100% concordance with the classification of Staphylococcus aureus resistance using culture. Discussion: Photoacoustics can be used to differentiate methicillin resistant and susceptible strains of bacteria from ocular infections. This method may be generalized to other bacterial species using appropriate bacteriophages and testing for resistance using other antibiotics.

Photoacoustic Flow Cytometry Using Functionalized Microspheres for Selective Detection of Bacteria.

Photoacoustic flow cytometry is a method to detect rare analytes in fluids. We developed photoacoustic flow cytometry to detect pathological cells in body fluids, such as circulating tumor cells or bacteria in blood. In order to induce specific optical absorption in bacteria, we use modified bacteriophage that precisely target bacterial species or subspecies for rapid identification. In order to reduce detection variability and to halt the lytic lifescycle that results in lysis of the bacteria, we attached dyed latex microspheres to the tail fibers of bacteriophage that retained the bacterial recognition binding sites. We tested these microsphere complexes using Salmonella enterica (Salmonella) and Escherichia coli (E. coli) bacteria and found robust and specific detection of targeted bacteria. In our work we used LT2, a strain of Salmonella, against K12, a strain of E. coli. Using Det7, a bacteriophage that binds to LT2 and not to K12, we detected an average of 109.3±9.0 of LT2 versus 2.0±1.7 of K12 using red microspheres and 86.7±13.2 of LT2 versus 0.3±0.6 of K12 using blue microspheres. These results confirmed our ability to selectively detect bacterial species using photoacoustic flow cytometry.

Photoacoustic discrimination of antibiotic-resistant and sensitive Staphylococcus aureus isolates.

OBJECTIVES: Bacteremia is a serious and potentially lethal condition. Staphylococcus aureus is a leading cause of bacteremia and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Compared to methicillin-sensitive S. aureus, MRSA is more than twice as likely to be fatal. Furthermore, subpopulations of seemingly isogenic bacteria may exhibit a range of susceptibilities, often called heterogenous resistance. These heterogeneous antibiotic-resistant infections are often misdiagnosed as hospital-acquired secondary infections because there are no clinically used tests that can differentiate between homogeneous and heterogeneous antibiotic resistance. We describe the development and proof of concept of rapid bacterial identification using photoacoustic flow cytometry and labeled bacteriophages with the characterization and differentiation of heterogeneous antibiotic-resistant bacterial infections. METHODS: In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer and particles that absorb laser light create an acoustic response. Optically labeled bacteriophage are added to a bacterial mixture that flows through the photoacoustic chamber. The presence of target bacteria is determined by bound labeled phage which are detected photoacoustically. Incubation of bacterial samples in the presence and absence of the antibiotic daptomycin creates a difference in bacterial cell numbers that is quantified using photoacoustic flow cytometry. RESULTS: Four clinical isolates were tested in the presence and absence of daptomycin. Photoacoustic events for each isolate were recorded and compared to growth curves. Samples treated with daptomycin fell into three categories: resistant, susceptible, and heterogeneous resistant. CONCLUSIONS: Here we show a method to determine the presence of bacteria as a marker for bloodstream infection level and antibiotic sensitivity in less than 4 hours. Additionally, these results show an ability to identify heterogeneous resistant strains that are often misidentified.

Capture and Isolation of Circulating Melanoma Cells Using Photoacoustic Flowmetry.

Early detection of cancer has been a goal of cancer research in general and melanoma research in particular (Birnbaum et al., Lancet Glob Health 6:e885-e893, 2018; Alendar et al., Bosnian J Basic Med Sci 9:77-80, 2009). Early detection of metastasis has been targeted as pivotal to increasing survival rates (Menezes et al., Adv Cancer Res 132:1-44, 2016). Melanoma, though curable in its early stages, has a dramatic decrease in survival rates once metastasis has occurred (Sharma et al., Biotechnol Adv 36:1063-1078, 2018). The transition to metastasis is not well understood and is an area of increasing interest. Metastasis is always premeditated by the shedding of circulating tumor cells (CTCs) from the primary tumor. The ability to isolate rare CTCs from the bloodstream has led to a host of new targets and therapies for cancer (Micalizzi et al., Genes Dev 31:1827-1840, 2017). Detection of CTCs also allows for disease progression to be tracked in real time while eliminating the need to wait for additional tumors to grow. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify and quantify these cells in order to track disease progression. Additionally, these CMCs are captured and isolated allowing for future analysis such as RNA-Seq or microarray analysis.

Predicting Metastasis in Melanoma by Enumerating Circulating Tumor Cells Using Photoacoustic Flow Cytometry.

BACKGROUND AND OBJECTIVES: Enumerating circulating tumor cells has been used as a method of monitoring progression of various cancers. Various methods for detecting circulating melanoma cells (CMCs) have been reported, but none has had sufficient sensitivity to determine if the presence of rare CMCs in the blood of Stage I-III melanoma patients predicts if those patients eventually develop metastatic disease. STUDY DESIGN: We quantified CMCs in serial blood samples from 38 early stage melanoma patients to determine if CMC numbers predict development of metastatic melanoma. CMCs were enumerated using a photoacoustic flow cytometric detection system that uses a laser to induce high frequency acoustic signals in pigmented CMCs. RESULTS: We observed that detection of greater than 2 CMCs/ml of blood from patients with Stage I-III melanoma predicts metastatic disease. Of the 11 patients we studied who had two or fewer CMCs detected at all time points tested, none progressed to metastatic disease over a mean follow-up of 1288 days. In contrast, 18 of the 27 patients (67%) having more than 2 CMCs/ml at one or more time points progressed to metastatic disease over a mean follow-up of 850 days. CONCLUSIONS: Photoacoustic flow cytometry can detect rare CMCs in the blood of Stage I-III melanoma patients and detectionof these cells is predictive of subsequent development of metastatic disease. Lasers Surg. Med.

Photoacoustic detection of circulating melanoma cells in late stage patients.

Melanoma is the deadliest skin cancer and is responsible for over 7000 deaths in the US annually. The spread of cancer, or metastasis, is responsible for these deaths, as secondary tumors interrupt normal organ function. Circulating tumor cells, or those cells that spread throughout the body from the primary tumor, are thought to be responsible for metastasis. We developed an optical method, photoacoustic flow cytometry, in order to detect and enumerate circulating melanoma cells (CMCs) from blood samples of patients. We tested the blood of Stage IV melanoma patients to show the ability of the photoacoustic flow cytometer to detect these rare cells in blood. We then tested the system on archived blood samples from Stage III melanoma patients with known outcomes to determine if detection of CMCs can predict future metastasis. We detected between 0 and 66 CMCs in Stage IV patients. For the Stage III study, we found that of those samples with CMCs, 2 remained disease free and 5 developed metastasis. Of those without CMCs, 6 remained disease free and 1 developed metastasis. We believe that photoacoustic detection of CMCs provides valuable information for the prediction of metastasis and we postulate a system for more accurate prognosis.

Bacteriophage-mediated identification of bacteria using photoacoustic flow cytometry.

Infection with resistant bacteria has become an ever increasing problem in modern medical practice. Currently, broad spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take two to three days and is unable to provide quantitative information. To detect and quantify bacteria rapidly in blood samples, we designed a method using labeled bacteriophage in conjunction with photoacoustic flow cytometry (PAFC). PAFC is the generation of ultrasonic waves created by the absorption of laser light in particles under flow. Bacteriophage is a virus that infects bacteria and possesses the ability to discriminate bacterial surface antigens, allowing the bacteriophage to bind only to their target bacteria. Bacteria can be tagged with dyed phage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. We demonstrate that E. coli; can be detected and discriminated from Salmonella; using this method. Our goal is to develop a method to determine bacterial content in blood samples. We hope to develop this technology into future clinical use and decrease the time required to identify bacterial species from 3 to 4 days to less than 1 hour.

Role of branchiomotor neurons in controlling food intake of zebrafish larvae.

The physical act of eating or feeding involves the coordinated action of several organs like eyes and jaws, and associated neural networks. Moreover, the activity of the neural networks controlling jaw movements (branchiomotor circuits) is regulated by the visual, olfactory, gustatory and hypothalamic systems, which are largely well characterized at the physiological level. By contrast, the behavioral output of the branchiomotor circuits and the functional consequences of disruption of these circuits by abnormal neural development are poorly understood. To begin to address these questions, we sought to evaluate the feeding ability of zebrafish larvae, a direct output of the branchiomotor circuits, and developed a qualitative assay for measuring food intake in zebrafish larvae at 7 days post-fertilization. We validated the assay by examining the effects of ablating the branchiomotor neurons. Metronidazole-mediated ablation of nitroreductase-expressing branchiomotor neurons resulted in a predictable reduction in food intake without significantly affecting swimming ability, indicating that the assay is robust. Laser-mediated ablation of trigeminal motor neurons resulted in a significant decrease in food intake, indicating that the assay is sensitive. Importantly, in larvae of a genetic mutant with severe loss of branchiomotor neurons, food intake was abolished. These studies establish a foundation for dissecting the neural circuits driving a motor behavior essential for survival.

Detection and capture of breast cancer cells with photoacoustic flow cytometry.

According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis­the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems­significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

Capture and Isolation of Circulating Melanoma Cells Using Photoacoustic Flowmetry.

Circulating tumor cells (CTCs) are those cells that separate from a solid tumor and spread through the blood or lymphatic systems. While there are many open questions concerning the biology of CTCs, there is mounting evidence that some of these cells go on to create secondary tumors in distant organs, thus enabling metastatic disease. Detection of CTCs may have clinical impact by providing prognostic information. Furthermore, molecular and genetic analysis of CTCs may enable cancer biologists to answer questions about the metastatic process, such as whether these cells undergo epithelial-mesenchymal transition. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify, capture, and isolate these cells for further analysis.

Quantitative photoacoustics to measure single cell melanin production and nanoparticle attachment.

Photoacoustics can be used as a label-free spectroscopic method of identifying pigmented proteins and characterizing their intracellular concentration over time in a single living cell. The authors use a microscopic laser irradiation system with a 5 ns, Q-switched laser focused onto single cells in order to collect photoacoustic responses of melanoma cells from the HS936 cell line and gold nanoparticle labeled breast cancer cells from the T47D cell line. The volume averaged intracellular concentration of melanin is found to range from 29-270 mM for single melanoma cells and the number of gold nanoparticles (AuNP) is shown to range from 850-5900 AuNPs/cell. Additionally, the melanin production response to UV-A light stimulus is measured in four melanoma cells to find a mass production rate of 5.7 pg of melanin every 15 min.

Detection of circulating tumor cells by photoacoustic flowmetry.

Detection of circulating tumor cells (CTCs) in human blood and lymph systems has the potential to aid clinical decision making in the treatment of cancer (Cristofanilli et al. New Engl J Med 351:781-791, 2004; Check Cap Today 19:1.76-1.86, 2005; Braun and Naume J Clin Oncol 8:1623-1626, 2005). The presence of CTCs may signify the onset of metastasis, indicate relapse, or may be used to monitor disease progression. We built and tested a photoacoustic flowmetry system for detecting circulating melanoma cells (CMCs) by exploiting the broadband absorption spectrum of melanin within CMCs. The device was tested on cultured melanoma cells in saline suspension, melanoma cells spiked in human blood, and in a Stage IV melanoma patient. The device showed a detection threshold of a single pigmented melanoma cell from culture. Results show the potential to assay blood samples from healthy and metastatic patients for the presence of cancerous melanoma providing a method for cancer screening.

Photoacoustic measurement of refractive index of dye solutions and myoglobin for biosensing applications.

Current methods of determining the refractive index of chemicals and materials, such as ellipsometry and reflectometry, are limited by their inability to analyze highly absorbing or highly transparent materials, as well as the required prior knowledge of the sample thickness and estimated refractive index. Here, we present a method of determining the refractive index of solutions using the photoacoustic effect. We show that a photoacoustic refractometer can analyze highly absorbing dye samples to within 0.006 refractive index units of a handheld optical refractometer. Further, we use myoglobin, an early non-invasive biomarker for malignant hyperthermia, as a proof of concept that this technique is applicable for use as a medical diagnostic. Comparison of the speed, cost, simplicity, and accuracy of the techniques shows that this photoacoustic method is well-suited for optically complex systems.

Nanoparticle mediated thermal ablation of breast cancer cells using a nanosecond pulsed electric field.

In the past, ablation of cancer cells using radiofrequency heating techniques has been demonstrated, but the current methodology has many flaws, including inconsistent tumor ablation and significant ablation of normal cells. Other researchers have begun to develop a treatment that is more selective for cancer cells using metallic nanoparticles and constant electric field exposure. In these studies, cell necrosis is induced by heating antibody functionalized metallic nanoparticles attached to cancer cells. Our approach to studying this phenomenon is to use similarly functionalized metallic nanoparticles that are specific for the T47D breast cancer cell line, exposing these nanoparticle cell conjugates to a nanosecond pulsed electric field. Using fluorescent, polystyrene-coated, iron-oxide nanoparticles, the results of our pilot study indicated that we were able to ablate up to approximately 80% of the cells using 60 ns pulses in increasing numbers of pulses and up to approximately 90% of the cells using 300 ns pulses in increasing numbers of pulses. These quantities of ablated cells were achieved using a cumulative exposure time 6 orders of magnitude less than most in vitro constant electric field studies.

Photoacoustic spectroscopy of ß-hematin.

Malaria affects over 200 million individuals annually, resulting in 800,000 fatalities. Current tests use blood smears and can only detect the disease when 0.1-1% of blood cells are infected. We are investigating the use of photoacoustic flowmetry to sense as few as one infected cell among 10 million or more normal blood cells, thus diagnosing infection before patients become symptomatic. Photoacoustic flowmetry is similar to conventional flow cytometry, except that rare cells are targeted by nanosecond laser pulses to induce ultrasonic responses. This system has been used to detect single melanoma cells in 10 ml of blood. Our objective is to apply photoacoustic flowmetry to detection of the malaria pigment hemozoin, which is a byproduct of parasite-digested hemoglobin in the blood. However, hemozoin is difficult to purify in quantities greater than a milligram, so a synthetic analog, known as ß-hematin was derived from porcine haemin. The specific purpose of this study is to establish the efficacy of using ß-hematin, rather than hemozoin, for photoacoustic measurements. We characterized ß-hematin using UV-vis spectroscopy, TEM, and FTIR, then tested the effects of laser irradiation on the synthetic product. We finally determined its absorption spectrum using photoacoustic excitation. UV-vis spectroscopy verified that ß-hematin was distinctly different from its precursor. TEM analysis confirmed its previously established nanorod shape, and comparison of the FTIR results with published spectroscopy data showed that our product had the distinctive absorbance peaks at 1661 and 1206 cm(-1). Also, our research indicated that prolonged irradiation dramatically alters the physical and optical properties of the ß-hematin, resulting in increased absorption at shorter wavelengths. Nevertheless, the photoacoustic absorption spectrum mimicked that generated by UV-vis spectroscopy, which confirms the accuracy of the photoacoustic method and strongly suggests that photoacoustic flowmetry may be used as a tool for diagnosis of malaria infection.

Total internal reflection photoacoustic spectroscopy for the detection of ß-hematin.

Evanescent field sensing methods are currently used to detect many different types of disease markers and biologically important chemicals such as the HER2 breast cancer receptor. Hinoue et al. used Total Internal Reflection Photoacoustic Spectroscopy (TIRPAS) as a method of using the evanescent field to detect an optically opaque dye at a sample interface. Although their methods were successful at detecting dyes, the results at that time did not show a very practical spectroscopic technique, which was due to the less than typical sensitivity of TIRPAS as a spectroscopy modality given the low power (≈ 1 to 2 W) lasers being used. Contrarily, we have used an Nd:YAG laser with a five nanosecond pulse that gives peak power of 1 MW coupled with the TIRPAS system to increase the sensitivity of this technique for biological material sensing. All efforts were focused on the eventual detection of the optically absorbing material, hemozoin, which is created as a byproduct of a malarial infection in blood. We used an optically analogous material, ß-hematin, to determine the potential for detection in the TIRPAS system. In addition, four properties which control the sensitivity were investigated to increase understanding about the sensor's function as a biosensing method.

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow.

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastatic melanoma cells in blood.

Detection and isolation of circulating melanoma cells using photoacoustic flowmetry.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors(1,2,3). CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient's response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)(4,5). This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid(6,7). PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.