Spatiotemporal Tracking of Brain-Tumor-Associated Myeloid Cells in Vivo through Optical Coherence Tomography with Plasmonic Labeling and Speckle Modulation.

By their nature, tumors pose a set of profound challenges to the immune system with respect to cellular recognition and response coordination. Recent research indicates that leukocyte subpopulations, especially tumor-associated macrophages (TAMs), can exert substantial influence on the efficacy of various cancer immunotherapy treatment strategies. To better study and understand the roles of TAMs in determining immunotherapeutic outcomes, significant technical challenges associated with dynamically monitoring single cells of interest in relevant live animal models of solid tumors must be overcome. However, imaging techniques with the requisite combination of spatiotemporal resolution, cell-specific contrast, and sufficient signal-to-noise at increasing depths in tissue are exceedingly limited. Here we describe a method to enable high-resolution, wide-field, longitudinal imaging of TAMs based on speckle-modulating optical coherence tomography (SM-OCT) and spectral scattering from an optimized contrast agent. The approach's improvements to OCT detection sensitivity and noise reduction enabled high-resolution OCT-based observation of individual cells of a specific host lineage in live animals. We found that large gold nanorods (LGNRs) that exhibit a narrow-band, enhanced scattering cross-section can selectively label TAMs and activate microglia in an in vivo orthotopic murine model of glioblastoma multiforme. We demonstrated near real-time tracking of the migration of cells within these myeloid subpopulations. The intrinsic spatiotemporal resolution, imaging depth, and contrast sensitivity reported herein may facilitate detailed studies of the fundamental behaviors of TAMs and other leukocytes at the single-cell level in vivo, including intratumoral distribution heterogeneity and roles in modulating cancer proliferation.

Speckle modulation enables high-resolution wide-field human brain tumor margin detection and in vivo murine neuroimaging.

Current in vivo neuroimaging techniques provide limited field of view or spatial resolution and often require exogenous contrast. These limitations prohibit detailed structural imaging across wide fields of view and hinder intraoperative tumor margin detection. Here we present a novel neuroimaging technique, speckle-modulating optical coherence tomography (SM-OCT), which allows us to image the brains of live mice and ex vivo human samples with unprecedented resolution and wide field of view using only endogenous contrast. The increased visibility provided by speckle elimination reveals white matter fascicles and cortical layer architecture in brains of live mice. To our knowledge, the data reported herein represents the highest resolution imaging of murine white matter structure achieved in vivo across a wide field of view of several millimeters. When applied to an orthotopic murine glioblastoma xenograft model, SM-OCT readily identifies brain tumor margins with resolution of approximately 10 µm. SM-OCT of ex vivo human temporal lobe tissue reveals fine structures including cortical layers and myelinated axons. Finally, when applied to an ex vivo sample of a low-grade glioma resection margin, SM-OCT is able to resolve the brain tumor margin. Based on these findings, SM-OCT represents a novel approach for intraoperative tumor margin detection and in vivo neuroimaging.

Real-Time Detection of Circulating Tumor Cells in Living Animals Using Functionalized Large Gold Nanorods.

Optical coherence tomography (OCT) can be utilized with significant speckle reduction techniques and highly scattering contrast agents for non-invasive, contrast-enhanced imaging of living tissues at the cellular scale. The advantages of reduced speckle noise and improved targeted contrast can be harnessed to track objects as small as 2 µm in vivo, which enables applications for cell tracking and quantification in living subjects. Here we demonstrate the use of large gold nanorods as contrast agents for detecting individual micron-sized polystyrene beads and single myeloma cells in blood circulation using speckle-modulating OCT. This report marks the first time that OCT has been used to detect individual cells within blood in vivo. This technical capability unlocks exciting opportunities for dynamic detection and quantification of tumor cells circulating in living subjects.

Optimization of the Trade-Off Between Speckle Reduction and Axial Resolution in Frequency Compounding.

We measured the reduction of speckle by frequency compounding using Gaussian pulses, which have the least time-bandwidth product. The experimental results obtained from a tissue mimicking phantom agree quantitatively with numerical simulations of randomly distributed point scatterers. For a fixed axial resolution, the amount of speckle reduction is found to approach a maximum as the number of bands increases while the total spectral range that they cover is kept constant. An analytical solution of the maximal speckle reduction is derived and shows that the maximum improves approximately as the inverse square root of the Gaussian pulse bandwidth. Since the axial resolution is proportional to the inverse of the pulse bandwidth, an optimized trade-off between speckle reduction and axial resolution is obtained. Considerations for the applications of the optimized trade-off are discussed.

Gold Nanoprisms as Optical Coherence Tomography Contrast Agents in the Second Near-Infrared Window for Enhanced Angiography in Live Animals.

Optical coherence tomography angiography (OCTA) is an important tool for investigating vascular networks and microcirculation in living tissue. Traditional OCTA detects blood vessels via intravascular dynamic scattering signals derived from the movements of red blood cells (RBCs). However, the low hematocrit and long latency between RBCs in capillaries make these OCTA signals discontinuous, leading to incomplete mapping of the vascular networks. OCTA imaging of microvascular circulation is particularly challenging in tumors due to the abnormally slow blood flow in angiogenic tumor vessels and strong attenuation of light by tumor tissue. Here, we demonstrate in vivo that gold nanoprisms (GNPRs) can be used as OCT contrast agents working in the second near-infrared window, significantly enhancing the dynamic scattering signals in microvessels and improving the sensitivity of OCTA in skin tissue and melanoma tumors in live mice. With GNPRs as contrast agents, the postinjection OCT angiograms showed 41 and 59% more microvasculature than preinjection angiograms in healthy mouse skin and melanoma tumors, respectively. By enabling better characterization of microvascular circulation in vivo, GNPR-enhanced OCTA could lead to better understanding of vascular functions during pathological conditions, more accurate measurements of therapeutic response, and improved patient prognoses.

Erratum: Speckle-modulating optical coherence tomography in living mice and humans.

This corrects the article DOI: 10.1038/ncomms15845.

Speckle-modulating optical coherence tomography in living mice and humans.

Optical coherence tomography (OCT) is a powerful biomedical imaging technology that relies on the coherent detection of backscattered light to image tissue morphology in vivo. As a consequence, OCT is susceptible to coherent noise (speckle noise), which imposes significant limitations on its diagnostic capabilities. Here we show speckle-modulating OCT (SM-OCT), a method based purely on light manipulation that virtually eliminates speckle noise originating from a sample. SM-OCT accomplishes this by creating and averaging an unlimited number of scans with uncorrelated speckle patterns without compromising spatial resolution. Using SM-OCT, we reveal small structures in the tissues of living animals, such as the inner stromal structure of a live mouse cornea, the fine structures inside the mouse pinna, and sweat ducts and Meissner's corpuscle in the human fingertip skin-features that are otherwise obscured by speckle noise when using conventional OCT or OCT with current state of the art speckle reduction methods.

Multimodal assessment of SERS nanoparticle biodistribution post ingestion reveals new potential for clinical translation of Raman imaging.

Despite extensive research and development, new nano-based diagnostic contrast agents have faced major barriers in gaining regulatory approval due to their potential systemic toxicity and prolonged retention in vital organs. Here we use five independent biodistribution techniques to demonstrate that oral ingestion of one such agent, gold-silica Raman nanoparticles, results in complete clearance with no systemic toxicity in living mice. The oral delivery mimics topical administration to the oral cavity and gastrointestinal (GI) tract as an alternative to intravenous injection. Biodistribution and clearance profiles of orally (OR) vs. intravenously (IV) administered Raman nanoparticles were assayed over the course of 48 h. Mice given either an IV or oral dose of Raman nanoparticles radiolabeled with approximately 100 µCi (3.7MBq) of 64Cu were imaged with dynamic microPET immediately post nanoparticle administration. Static microPET images were also acquired at 2 h, 5 h, 24 h and 48 h. Mice were sacrificed post imaging and various analyses were performed on the excised organs to determine nanoparticle localization. The results from microPET imaging, gamma counting, Raman imaging, ICP-MS, and hyperspectral imaging of tissue sections all correlated to reveal no evidence of systemic distribution of Raman nanoparticles after oral administration and complete clearance from the GI tract within 24 h. Paired with the unique signals and multiplexing potential of Raman nanoparticles, this approach holds great promise for realizing targeted imaging of tumors and dysplastic tissues within the oral cavity and GI-tract. Moreover, these results suggest a viable path for the first translation of high-sensitivity Raman contrast imaging into clinical practice.

In Vivo Molecular Optical Coherence Tomography of Lymphatic Vessel Endothelial Hyaluronan Receptors.

Optical Coherence Tomography (OCT) imaging of living subjects offers increased depth of penetration while maintaining high spatial resolution when compared to other optical microscopy techniques. However, since most protein biomarkers do not exhibit inherent contrast detectable by OCT, exogenous contrast agents must be employed for imaging specific cellular biomarkers of interest. While a number of OCT contrast agents have been previously studied, demonstrations of molecular targeting with such agents in live animals have been historically challenging and notably limited in success. Here we demonstrate for the first time that microbeads (µBs) can be used as contrast agents to target cellular biomarkers in lymphatic vessels and can be detected by OCT using a phase variance algorithm. This molecular OCT method enables in vivo imaging of the expression profiles of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a biomarker that plays crucial roles in inflammation and tumor metastasis. In vivo OCT imaging of LVYE-1 showed that the biomarker was significantly down-regulated during inflammation induced by acute contact hypersensitivity (CHS). Our work demonstrated a powerful molecular imaging tool that can be used for high resolution studies of lymphatic function and dynamics in models of inflammation, tumor development, and other lymphatic diseases.

A hyperspectral method to assay the microphysiological fates of nanomaterials in histological samples.

Nanoparticles are used extensively as biomedical imaging probes and potential therapeutic agents. As new particles are developed and tested in vivo, it is critical to characterize their biodistribution profiles. We demonstrate a new method that uses adaptive algorithms for the analysis of hyperspectral dark-field images to study the interactions between tissues and administered nanoparticles. This non-destructive technique quantitatively identifies particles in ex vivo tissue sections and enables detailed observations of accumulation patterns arising from organ-specific clearance mechanisms, particle size, and the molecular specificity of nanoparticle surface coatings. Unlike nanoparticle uptake studies with electron microscopy, this method is tractable for imaging large fields of view. Adaptive hyperspectral image analysis achieves excellent detection sensitivity and specificity and is capable of identifying single nanoparticles. Using this method, we collected the first data on the sub-organ distribution of several types of gold nanoparticles in mice and observed localization patterns in tumors.

High-resolution contrast-enhanced optical coherence tomography in mice retinae.

Optical coherence tomography (OCT) is a noninvasive interferometric imaging modality providing anatomical information at depths of millimeters and a resolution of micrometers. Conventional OCT images limit our knowledge to anatomical structures alone, without any contrast enhancement. Therefore, here we have, for the first time, optimized an OCT-based contrast-enhanced imaging system for imaging single cells and blood vessels in vivo inside the living mouse retina at subnanomolar sensitivity. We used bioconjugated gold nanorods (GNRs) as exogenous OCT contrast agents. Specifically, we used anti-mouse CD45 coated GNRs to label mouse leukocytes and mPEG-coated GNRs to determine sensitivity of GNR detection in vivo inside mice retinae. We corroborated OCT observations with hyperspectral dark-field microscopy of formalin-fixed histological sections. Our results show that mouse leukocytes that otherwise do not produce OCT contrast can be labeled with GNRs leading to significant OCT intensity equivalent to a 0.5 nM GNR solution. Furthermore, GNRs injected intravenously can be detected inside retinal blood vessels at a sensitivity of ∼0.5 nM, and GNR-labeled cells injected intravenously can be detected inside retinal capillaries by enhanced OCT contrast. We envision the unprecedented resolution and sensitivity of functionalized GNRs coupled with OCT to be adopted for longitudinal studies of retinal disorders.

Contrast-enhanced optical coherence tomography with picomolar sensitivity for functional in vivo imaging.

Optical Coherence Tomography (OCT) enables real-time imaging of living tissues at cell-scale resolution over millimeters in three dimensions. Despite these advantages, functional biological studies with OCT have been limited by a lack of exogenous contrast agents that can be distinguished from tissue. Here we report an approach to functional OCT imaging that implements custom algorithms to spectrally identify unique contrast agents: large gold nanorods (LGNRs). LGNRs exhibit 110-fold greater spectral signal per particle than conventional GNRs, which enables detection of individual LGNRs in water and concentrations as low as 250 pM in the circulation of living mice. This translates to ~40 particles per imaging voxel in vivo. Unlike previous implementations of OCT spectral detection, the methods described herein adaptively compensate for depth and processing artifacts on a per sample basis. Collectively, these methods enable high-quality noninvasive contrast-enhanced imaging of OCT in living subjects, including detection of tumor microvasculature at twice the depth achievable with conventional OCT. Additionally, multiplexed detection of spectrally-distinct LGNRs was demonstrated to observe discrete patterns of lymphatic drainage and identify individual lymphangions and lymphatic valve functional states. These capabilities provide a powerful platform for molecular imaging and characterization of tissue noninvasively at cellular resolution, called MOZART.

Quantitative contrast-enhanced optical coherence tomography.

We have developed a model to accurately quantify the signals produced by exogenous scattering agents used for contrast-enhanced Optical Coherence Tomography (OCT). This model predicts distinct concentration-dependent signal trends that arise from the underlying physics of OCT detection. Accordingly, we show that real scattering particles can be described as simplified ideal scatterers with modified scattering intensity and concentration. The relation between OCT signal and particle concentration is approximately linear at concentrations lower than 0.8 particle per imaging voxel. However, at higher concentrations, interference effects cause signal to increase with a square root dependence on the number of particles within a voxel. Finally, high particle concentrations cause enough light attenuation to saturate the detected signal. Predictions were validated by comparison with measured OCT signals from gold nanorods (GNRs) prepared in water at concentrations ranging over five orders of magnitude (50 fM to 5 nM). In addition, we validated that our model accurately predicts the signal responses of GNRs in highly heterogeneous scattering environments including whole blood and living animals. By enabling particle quantification, this work provides a valuable tool for current and future contrast-enhanced in vivo OCT studies. More generally, the model described herein may inform the interpretation of detected signals in modalities that rely on coherence-based detection or are susceptible to interference effects.

Biofunctionalization of Large Gold Nanorods Realizes Ultrahigh-Sensitivity Optical Imaging Agents.

Gold nanorods (GNRs, ∼ 50 × 15 nm) have been used ubiquitously in biomedicine for their optical properties, and many methods of GNR biofunctionalization have been described. Recently, the synthesis of larger-than-usual GNRs (LGNRs, ∼ 100 × 30 nm) has been demonstrated. However, LGNRs have not been biofunctionalized and therefore remain absent from biomedical literature to date. Here we report the successful biofunctionalization of LGNRs, which produces highly stable particles that exhibit a narrow spectral peak (FWHM ∼100 nm). We further demonstrated that functionalized LGNRs can be used as highly sensitive scattering contrast agents by detecting individual LGNRs in clear liquids. Owing to their increased optical cross sections, we found that LGNRs exhibited up to 32-fold greater backscattering than conventional GNRs. We leveraged these enhanced optical properties to detect LGNRs in the vasculature of live tumor-bearing mice. With LGNR contrast enhancement, we were able to visualize tumor blood vessels at depths that were otherwise undetectable. We expect that the particles reported herein will enable immediate sensitivity improvements in a wide array of biomedical imaging and sensing techniques that rely on conventional GNRs.