In vivo Raman spectroscopy monitors cervical change during labor.

BACKGROUND: Biochemical cervical change during labor is not well understood, in part, because of a dearth of technologies capable of safely probing the pregnant cervix in vivo. The need for such a technology is 2-fold: (1) to gain a mechanistic understanding of the cervical ripening and dilation process and (2) to provide an objective method for evaluating the cervical state to guide clinical decision-making. Raman spectroscopy demonstrates the potential to meet this need, as it is a noninvasive optical technique that can sensitively detect alterations in tissue components, such as extracellular matrix proteins, lipids, nucleic acids, and blood, which have been previously established to change during the cervical remodeling process. OBJECTIVE: We sought to demonstrate that Raman spectroscopy can longitudinally monitor biochemical changes in the laboring cervix to identify spectral markers of impending parturition. STUDY DESIGN: Overall, 30 pregnant participants undergoing either spontaneous or induced labor were recruited. The Raman spectra were acquired in vivo at 4-hour intervals throughout labor until rupture of membranes using a Raman system with a fiber-optic probe. Linear mixed-effects models were used to determine significant (P<.05) changes in peak intensities or peak ratios as a function of time to delivery in the study population. A nonnegative least-squares biochemical model was used to extract the changing contributions of specific molecule classes over time. RESULTS: We detected multiple biochemical changes during labor, including (1) significant decreases in Raman spectral features associated with collagen and other extracellular matrix proteins (P=.0054) attributed to collagen dispersion, (2) an increase in spectral features associated with blood (P=.0372), and (3) an increase in features indicative of lipid-based molecules (P=.0273). The nonnegative least-squares model revealed a decrease in collagen contribution with time to delivery, an increase in blood contribution, and a change in lipid contribution. CONCLUSION: Our findings have demonstrated that in vivo Raman spectroscopy is sensitive to multiple biochemical remodeling changes in the cervix during labor. Furthermore, in vivo Raman spectroscopy may be a valuable noninvasive tool for objectively evaluating the cervix to potentially guide clinical management of labor.

Monitoring uterine contractility in mice using a transcervical intrauterine pressure catheter.

In mouse models used to study parturition or pre-clinical therapeutic testing, measurement of uterine contractions is limited to either ex vivo isometric tension or operative intrauterine pressure (IUP). The goal of this study was to: (1) develop a method for transcervical insertion of a pressure catheter to measure in vivo intrauterine contractile pressure during mouse pregnancy, (2) determine whether this method can be utilized numerous times in a single mouse pregnancy without affecting the timing of delivery or fetal outcome and (3) compare the in vivo contractile activity between mouse models of term and preterm labor (PTL). Visualization of the cervix allowed intrauterine pressure catheter (IUPC) placement into anesthetized pregnant mice (plug = day 1, delivery = day 19.5). The amplitude, frequency, duration and area under the curve (AUC) of IUP was lowest on days 16-18, increased significantly (P < 0.05) on the morning of day 19 and reached maximal levels during by the afternoon of day 19 and into the intrapartum period. An AUC threshold of 2.77 mmHg discriminated between inactive labor (day 19 am) and active labor (day 19 pm and intrapartum period). Mice examined on a single vs every experimental timepoint did not have significantly different IUP, timing of delivery, offspring number or fetal/neonatal weight. The IUP was significantly greater in LPS-treated and RU486-treated mouse models of PTL compared to time-matched vehicle control mice. Intrapartum IUP was not significantly different between term and preterm mice. We conclude that utilization of a transcervical IUPC allows sensitive assessment of in vivo uterine contractile activity and labor progression in mouse models without the need for operative approaches.

In vivo Raman spectroscopy for biochemical monitoring of the human cervix throughout pregnancy.

BACKGROUND: The cervix must undergo significant biochemical remodeling to allow for successful parturition. This process is not fully understood, especially in instances of spontaneous preterm birth. In vivo Raman spectroscopy is an optical technique that can be used to investigate the biochemical composition of tissue longitudinally and noninvasively in human beings, and has been utilized to measure physiology and disease states in a variety of medical applications. OBJECTIVE: The purpose of this study is to measure in vivo Raman spectra of the cervix throughout pregnancy in women, and to identify biochemical markers that change with the preparation for delivery and postpartum repair. STUDY DESIGN: In all, 68 healthy pregnant women were recruited. Raman spectra were measured from the cervix of each patient monthly in the first and second trimesters, weekly in the third trimester, and at the 6-week postpartum visit. Raman spectra were measured using an in vivo Raman system with an optical fiber probe to excite the tissue with 785 nm light. A spectral model was developed to highlight spectral regions that undergo the most changes throughout pregnancy, which were subsequently used for identifying Raman peaks for further analysis. These peaks were analyzed longitudinally to determine if they underwent significant changes over the course of pregnancy (P < .05). Finally, 6 individual components that comprise key biochemical constituents of the human cervix were measured to extract their contributions in spectral changes throughout pregnancy using a linear combination method. Patient factors including body mass index and parity were included as variables in these analyses. RESULTS: Raman peaks indicative of extracellular matrix proteins (1248 and 1254 cm-1) significantly decreased (P < .05), while peaks corresponding to blood (1233 and 1563 cm-1) significantly increased (P < .0005) in a linear manner throughout pregnancy. In the postpartum cervix, significant increases in peaks corresponding to actin (1003, 1339, and 1657 cm-1) and cholesterol (1447 cm-1) were observed when compared to late gestation, while signatures from blood significantly decreased. Postpartum actin signals were significantly higher than early pregnancy, whereas extracellular matrix proteins and water signals were significantly lower than early weeks of gestation. Parity had a significant effect on blood and extracellular matrix protein signals, with nulliparous patients having significant increases in blood signals throughout pregnancy, and higher extracellular matrix protein signals in early pregnancy compared to patients with prior pregnancies. Body mass index significantly affected actin signal contribution, with low body mass index patients showing decreasing actin contribution throughout pregnancy and high body mass index patients demonstrating increasing actin signals. CONCLUSION: Raman spectroscopy was successfully used to biochemically monitor cervical remodeling in pregnant women during prenatal visits. This foundational study has demonstrated sensitivity to known biochemical dynamics that occur during cervical remodeling, and identified patient variables that have significant effects on Raman spectra throughout pregnancy. Raman spectroscopy has the potential to improve our understanding of cervical maturation, and be used as a noninvasive preterm birth risk assessment tool to reduce the incidence, morbidity, and mortality caused by preterm birth.

Dual excitation wavelength system for combined fingerprint and high wavenumber Raman spectroscopy.

A fiber optic probe-based Raman spectroscopy system using a single laser module with two excitation wavelengths, at 680 and 785 nm, has been developed for measuring the fingerprint and high wavenumber regions using a single detector. This system is simpler and less expensive than previously reported configurations of combined fingerprint and high wavenumber Raman systems, and its probe-based implementation facilitates numerous in vivo applications. The high wavenumber region of the Raman spectrum ranges from 2800-3800 cm-1 and contains valuable information corresponding to the molecular vibrations of proteins, lipids, and water, which is complimentary to the biochemical signatures found in the fingerprint region (800-1800 cm-1), which probes DNA, lipids, and proteins. The efficacy of the system is demonstrated by tracking changes in water content in tissue-mimicking phantoms, where Voigtian decomposition of the high wavenumber water peak revealed a correlation between the water content and type of water-tissue interactions in the samples. This dual wavelength system was then used for in vivo assessment of cervical remodeling during mouse pregnancy, a physiologic process with known changes in tissue hydration. The system shows that Raman spectroscopy is sensitive to changes in collagen content in the fingerprint region and hydration state in the high wavenumber region, which was verified using an ex vivo comparison of wet and dry weight. Simultaneous fingerprint and high wavenumber Raman spectroscopy will allow precise in vivo quantification of tissue water content in the high wavenumber region, paired with the high biochemical specificity of the fingerprint region.

Raman spectroscopy provides a noninvasive approach for determining biochemical composition of the pregnant cervix in vivo.

UNLABELLED: The molecular changes that occur with cervical remodelling during pregnancy are not completely understood. This study reviews Raman spectroscopy, an optical technique for detecting changes in the pregnant cervix, and reports preliminary studies on cervical remodelling in mice that suggest that the technique provides advantages over other methods. CONCLUSION: Raman spectroscopy is sensitive to biochemical changes in the pregnant cervix and has high potential as a tool for detecting premature cervical remodelling in pregnant women.

Low-cost augmented reality goggles enable precision fluorescence-guided cancer surgery.

Disparities in surgical outcomes often result from subjective than objective decisions dictated by surgical training, experience, and available resources. To improve outcomes, surgeons have adopted advancements in robotics, endoscopy, and intra-operative imaging including fluorescence-guided surgery (FGS), which highlight tumors in real-time without using ionizing radiation. However, like many medical innovations, technical, economic, and logistic challenges have hindered widespread adoption of FGS beyond high-resource centers. To overcome these impediments, we developed the fully-wearable and battery-powered fluorescence imaging augmented reality Raspberry Pi-based goggle system (FAR-Pi). Novel device design ensures distance-independent coalignment between real and augmented FAR-Pi views and offers higher spatial resolution, depth of focus, and fluorescence detection sensitivity than existing bulkier, pricier, and wall-powered technologies. When paired with pan-tumor targeting fluorescent agents such as LS301, FAR-Pi objectively identifies tumors in vivo. As an open-source, affordable, and adaptable system, FAR-Pi is poised to democratize access to FGS and improve health outcomes worldwide.

Development and evaluation of a wearable peripheral vascular compensation sensor in a swine model of hemorrhage.

Postpartum hemorrhage (PPH) is both the leading and most preventable cause of maternal mortality. PPH is currently diagnosed through visual estimation of blood loss or vital sign analysis of shock index (ratio of heart rate to systolic blood pressure). Visual assessment underestimates blood loss, particularly in the setting of internal bleeding, and compensatory mechanisms stabilize hemodynamics until hemorrhage is massive, beyond the point of pharmaceutical intervention. Quantitative monitoring of hemorrhage-induced compensatory processes, such as the constriction of peripheral vessels to shunt blood to the central organs, may provide an early alert for PPH. To this end, we developed a low-cost, wearable optical device that continuously monitors peripheral perfusion via laser speckle flow index (LSFI) to detect hemorrhage-induced peripheral vasoconstriction. The device was first tested using flow phantoms across a range of physiologically relevant flow rates and demonstrated a linear response. Subsequent testing occurred in swine hemorrhage studies (n=6) by placing the device on the posterior side of the swine's front hock and withdrawing blood from the femoral vein at a constant rate. Resuscitation with intravenous crystalloids followed the induced hemorrhage. The mean LSFI vs. percent estimated blood volume loss had an average correlation coefficient of -0.95 during the hemorrhage phase and 0.79 during resuscitation, both of which were superior to the performance of the shock index. With continued development, this noninvasive, low-cost, and reusable device has global potential to provide an early alert of PPH when low-cost and accessible management strategies are most effective, helping to reduce maternal morbidity and mortality from this largely preventable problem.

Development and evaluation of a wearable peripheral vascular compensation sensor in a swine model of hemorrhage.

Postpartum hemorrhage (PPH) is the leading and most preventable cause of maternal mortality, particularly in low-resource settings. PPH is currently diagnosed through visual estimation of blood loss or monitoring of vital signs. Visual assessment routinely underestimates blood loss beyond the point of pharmaceutical intervention. Quantitative monitoring of hemorrhage-induced compensatory processes, such as the constriction of peripheral vessels, may provide an early alert for PPH. To this end, we developed a low-cost, wearable optical device that continuously monitors peripheral perfusion via laser speckle flow index (LSFI) to detect hemorrhage-induced peripheral vasoconstriction. The measured LSFI signal produced a linear response in phantom models and a strong correlation coefficient with blood loss averaged across subjects (>0.9) in a large animal model, with superior performance to vital sign metrics.

Quantitative tumor depth determination using dual wavelength excitation fluorescence.

Quantifying solid tumor margins with fluorescence-guided surgery approaches is a challenge, particularly when using near infrared (NIR) wavelengths due to increased penetration depths. An NIR dual wavelength excitation fluorescence (DWEF) approach was developed that capitalizes on the wavelength-dependent attenuation of light in tissue to determine fluorophore depth. A portable dual wavelength excitation fluorescence imaging system was built and tested in parallel with an NIR tumor-targeting fluorophore in tissue mimicking phantoms, chicken tissue, and in vivo mouse models of breast cancer. The system showed high accuracy in all experiments. The low cost and simplicity of this approach make it ideal for clinical use.

Application driven assessment of probe designs for Raman spectroscopy.

In vivo Raman spectroscopy has been utilized for the non-invasive, non-destructive assessment of tissue pathophysiology for a variety of applications largely through the use of fiber optic probes to interface with samples of interest. Fiber optic probes can be designed to optimize the collection of Raman-scattered photons from application-dependent depths, and this critical consideration should be addressed when planning a study. Herein we investigate four distinct probe geometries for sensitivity to superficial and deep signals through a Monte Carlo model that incorporates Raman scattering and fluorescence. Experimental validation using biological tissues was performed to accurately recapitulate in vivo scenarios. Testing in biological tissues agreed with modeled results and revealed that microlens designs had slightly enhanced performance at shallow depths (< 1 mm), whereas all of the beampath-modified designs yielded more signal from deep within tissue. Simulation based on fluence maps generated using ray-tracing in the absence of optical scattering had drastically different results as a function of depth for each probe compared to the biological simulation. The contrast in simulation results between the non-scattering and biological tissue phantoms underscores the importance of considering the optical properties of a given application when designing a fiber optic probe. The model presented here can be easily extended for optimization of entirely novel probe designs prior to fabrication, reducing time and cost while improving data quality.

Repurposing Molecular Imaging and Sensing for Cancer Image-Guided Surgery.

Gone are the days when medical imaging was used primarily to visualize anatomic structures. The emergence of molecular imaging (MI), championed by radiolabeled 18F-FDG PET, has expanded the information content derived from imaging to include pathophysiologic and molecular processes. Cancer imaging, in particular, has leveraged advances in MI agents and technology to improve the accuracy of tumor detection, interrogate tumor heterogeneity, monitor treatment response, focus surgical resection, and enable image-guided biopsy. Surgeons are actively latching on to the incredible opportunities provided by medical imaging for preoperative planning, intraoperative guidance, and postoperative monitoring. From label-free techniques to enabling cancer-selective imaging agents, image-guided surgery provides surgical oncologists and interventional radiologists both macroscopic and microscopic views of cancer in the operating room. This review highlights the current state of MI and sensing approaches available for surgical guidance. Salient features of nuclear, optical, and multimodal approaches will be discussed, including their strengths, limitations, and clinical applications. To address the increasing complexity and diversity of methods available today, this review provides a framework to identify a contrast mechanism, suitable modality, and device. Emerging low-cost, portable, and user-friendly imaging systems make the case for adopting some of these technologies as the global standard of care in surgical practice.

Focal dynamic thermal imaging for label-free high-resolution characterization of materials and tissue heterogeneity.

Evolution from static to dynamic label-free thermal imaging has improved bulk tissue characterization, but fails to capture subtle thermal properties in heterogeneous systems. Here, we report a label-free, high speed, and high-resolution platform technology, focal dynamic thermal imaging (FDTI), for delineating material patterns and tissue heterogeneity. Stimulation of focal regions of thermally responsive systems with a narrow beam, low power, and low cost 405 nm laser perturbs the thermal equilibrium. Capturing the dynamic response of 3D printed phantoms, ex vivo biological tissue, and in vivo mouse and rat models of cancer with a thermal camera reveals material heterogeneity and delineates diseased from healthy tissue. The intuitive and non-contact FDTI method allows for rapid interrogation of suspicious lesions and longitudinal changes in tissue heterogeneity with high-resolution and large field of view. Portable FDTI holds promise as a clinical tool for capturing subtle differences in heterogeneity between malignant, benign, and inflamed tissue.

Development of a visually guided Raman spectroscopy probe for cervical assessment during pregnancy.

Preterm birth (PTB) is the leading cause of neonatal death, however, accurate prediction methods do not exist. Detection of early changes in the cervix, an organ that biochemically remodels to deliver the fetus, has potential to predict PTB risk. Researchers have employed light-based methods to monitor biochemical changes in the cervix during pregnancy, however, these approaches required patients to undergo a speculum examination which many patients find uncomfortable and is not standard practice during prenatal care. Herein, a visually guided optical probe is presented that measures the cervix via introduction by bimanual examination, a procedure that is commonly performed during prenatal visits and labor for tactile monitoring of the cervix. The device incorporates a Raman spectroscopy probe for biochemical monitoring and a camera for visualizing measurement location to ensure it is void of cervical mucus and blood. This probe was tested in 15 patients receiving obstetric and gynecological care, and results acquired with and without a speculum revealed similar spectra, demonstrating that the visually guided probe conserved data quality. Additionally, the majority of patients reported reduced discomfort from the device. In summary, the visual guidance probe successfully measured the cervix while integrating with standard prenatal care, reducing a barrier in clinical translation.

Raman microspectroscopy differentiates perinatal pathogens on ex vivo infected human fetal membrane tissues.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a major cause of chorioamnionitis and neonatal sepsis. This study evaluates Raman spectroscopy (RS) to identify spectral characteristics of infection and differentiate GBS from Escherichia coli and Staphylococcus aureus during ex vivo infection of human fetal membrane tissues. Unique spectral features were identified from colonies grown on agar and infected fetal membrane tissues. Multinomial logistic regression analysis accurately identified GBS infected tissues with 100.0% sensitivity and 88.9% specificity. Together, these findings support further investigation into the use of RS as an emerging microbiologic diagnostic tool and intrapartum screening test for GBS carriage.

Diagnosis of immunomarkers in vivo via multiplexed surface enhanced Raman spectroscopy with gold nanostars.

In this work, we demonstrate the targeted diagnosis of immunomarker programmed death ligand 1 (PD-L1) and simultaneous detection of epidermal growth factor receptor (EGFR) in breast cancer tumors in vivo using gold nanostars (AuNS) with multiplexed surface enhanced Raman spectroscopy (SERS). Real-time longitudinal tracking with SERS demonstrated maximum accumulation of AuNS occurred 6 h post intravenous (IV) delivery, enabling detection of both biomarkers simultaneously. Raman signal correlating to both PD-L1 and EGFR decreased by ∼30% in control tumors where receptors were pre-blocked prior to AuNS delivery, indicating both the sensitivity and specificity of SERS in distinguishing tumors with different levels of PD-L1 and EGFR expression. Our in vivo study was combined with the first demonstration of ex vivo SERS spatial maps of whole tumor lesions that provided both a qualitative and quantitative assessment of biomarker status with near cellular-level resolution. High resolution SERS maps also provided an overview of AuNS distribution in tumors which correlated well with the vascular density. Mass spectrometry showed AuNS accumulation in tumor and liver, and clearance via spleen, and electron microscopy revealed AuNS were endocytosed in tumors, Kupffer cells in the liver, and macrophages in the spleen. This study demonstrates that SERS-based diagnosis mediated by AuNS provides an accurate measure of multiple biomarkers both in vivo and ex vivo, which will ultimately enable a clinically-translatable platform for patient-tailored immunotherapies and combination treatments.

Drug-Free ROS Sponge Polymeric Microspheres Reduce Tissue Damage from Ischemic and Mechanical Injury.

The inherent antioxidant function of poly(propylene sulfide) (PPS) microspheres (MS) was dissected for different reactive oxygen species (ROS), and therapeutic benefits of PPS-MS were explored in models of diabetic peripheral arterial disease (PAD) and mechanically induced post-traumatic osteoarthritis (PTOA). PPS-MS (∼1 µm diameter) significantly scavenged hydrogen peroxide (H2O2), hypochlorite, and peroxynitrite but not superoxide in vitro in cell-free and cell-based assays. Elevated ROS levels (specifically H2O2) were confirmed in both a mouse model of diabetic PAD and in a mouse model of PTOA, with greater than 5- and 2-fold increases in H2O2, respectively. PPS-MS treatment functionally improved recovery from hind limb ischemia based on ∼15-25% increases in hemoglobin saturation and perfusion in the footpads as well as earlier remodeling of vessels in the proximal limb. In the PTOA model, PPS-MS reduced matrix metalloproteinase (MMP) activity by 30% and mitigated the resultant articular cartilage damage. These results suggest that local delivery of PPS-MS at sites of injury-induced inflammation improves the vascular response to ischemic injury in the setting of chronic hyperglycemia and reduces articular cartilage destruction following joint trauma. These results motivate further exploration of PPS as a stand-alone, locally sustained antioxidant therapy and as a material for microsphere-based, sustained local drug delivery to inflamed tissues at risk of ROS damage.

In vivo Raman spectral analysis of impaired cervical remodeling in a mouse model of delayed parturition.

Monitoring cervical structure and composition during pregnancy has high potential for prediction of preterm birth (PTB), a problem affecting 15 million newborns annually. We use in vivo Raman spectroscopy, a label-free, light-based method that provides a molecular fingerprint to non-invasively investigate normal and impaired cervical remodeling. Prostaglandins stimulate uterine contractions and are clinically used for cervical ripening during pregnancy. Deletion of cyclooxygenase-1 (Cox-1), an enzyme involved in production of these prostaglandins, results in delayed parturition in mice. Contrary to expectation, Cox-1 null mice displayed normal uterine contractility; therefore, this study sought to determine whether cervical changes could explain the parturition differences in Cox-1 null mice and gestation-matched wild type (WT) controls. Raman spectral changes related to extracellular matrix proteins, lipids, and nucleic acids were tracked over pregnancy and found to be significantly delayed in Cox-1 null mice at term. A cervical basis for the parturition delay was confirmed by other ex vivo tests including decreased tissue distensibility, hydration, and elevated progesterone levels in the Cox-1 null mice at term. In conclusion, in vivo Raman spectroscopy non-invasively detected abnormal remodeling in the Cox-1 null mouse, and clearly demonstrated that the cervix plays a key role in their delayed parturition.

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.