Nanoscale materials are routinely developed, characterized, and evaluated as diagnostic and therapeutic agents for disease treatment in humans. However, the size and composition of many such agents result in poor clearance profiles within biological tissues, thereby posing profound challenges to translational clinical applications. Herein, we present a hyperspectral imaging technique capable of quantifying plasmonic nanoparticle biodistribution with single particle limit of detection and microanatomical detail. Using this method, we find that, although intravenous administration of plasmonic nanoparticles remains largely infeasible from a biodistribution perspective, alternate routes relevant to treatment of oral and gastrointestinal diseases are within translational reach.
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. This is the first demonstration that nanoparticle-based OCT contrast agent works in vivo in the second near infrared window, which allows deeper imaging depth by OCT. With GNPRs as contrast agents, the post-injection OCT angiograms showed 41% and 59% more microvasculature than pre-injection 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.
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, known as 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, which can remove speckle noise, including noise originating from sample multiple back-scattering. SM-OCT accomplishes this by creating and averaging an unlimited number of scans with uncorrelated speckle patterns, without compromising spatial resolution. The uncorrelated speckle patterns are created by scrambling the phase of the light with sub-resolution features using a moving ground-glass diffuser in the optical path of the sample arm. This method can be implemented in existing OCTs as a relatively low-cost add-on. SM-OCT speckle statistics follow the expected decrease in speckle contrast as the number of averaged scans increases. Within a scattering phantom, SM-OCT provides a 2.5-fold increase in effective resolution compared to conventional OCT. 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. Our results indicate that SM-OCT has the potential to improve the current diagnostic and intra-operative capabilities of OCT.
Optical Coherence Tomography (OCT) imaging of living subjects offers millimeters depth of penetration into tissue while maintaining high spatial resolution. However, because most molecular biomarkers do not produce inherent OCT contrast signals, exogenous contrast agents must be employed to achieve molecular imaging. Here we demonstrate that microbeads (μBs) can be used as effective contrast agents to target cellular biomarkers in lymphatic vessels and can be detected by OCT using a phase variance algorithm. We applied this technique to image the molecular dynamics of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) in vivo, which showed significant down-regulation during tissue inflammation.
Optical Coherence Tomography (OCT) is well-suited to study in vivo dynamics of blood circulation and lymphatic flow because of the technique’s combination of rapid image acquisition, micron spatial resolution, and penetration depth in turbid tissues. However, OCT has been historically constrained by a dearth of contrast agents that are readily distinguished from the strong scattering intrinsic to biological tissues. In this study, we demonstrate large gold nanorods (LGNRs) as optimized contrast agents for OCT. LGNRs produce 32-fold greater backscattering than GNRs previously tested for contrast-enhanced OCT. Furthermore, LGNRs exhibit 110-fold stronger spectral signal than conventional GNRs when coupled with custom spectral detection algorithms. This signal enhancement enables picomolar OCT detection sensitivity in vivo and single-particle detection against optically-clear backgrounds. Moreover, the ability to synthesize LGNRs with tunable spectral peaks provides a viable platform for multiplexed imaging studies. To explore the advantages of LGNRs as OCT contrast agents, we implemented them for noninvasive 3D imaging of tumor blood supply and active lymphatic drainage in mice. Spectral detection of LGNRs enabled 100% improvement in imaging depth for detecting microvasculature (vessels ∼ 20 μm in diameter) in U87MG glioblastoma xenografts in mice pinnae. We also demonstrated our approach’s ability to map the spatial dependence of lymph drainage and flow directionality within lymphatic capillaries. Using LGNRs with distinct spectra, we further identified the functional states of individual lymphatic valves in vivo. Thus, this approach provides a powerful new platform for functional imaging that may be extended for future molecular imaging studies with OCT.
KEYWORDS: Optical coherence tomography, Signal detection, Particles, Scattering, Signal attenuation, Ultrasonography, Speckle, Blood, In vivo imaging, Mie scattering
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 coherence-based 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 particles 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 5nM). 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 be applied for detected signals in other modalities that rely on coherence-based detection or are susceptible to interference effects, most notably medical ultrasound. Thus, our model may enable quantitative interpretation of ultrasound contrast agents including gas-filled microbubbles.
Nanoparticles have been explored extensively as potential biomedical imaging and therapeutic agents. One critical aspect of in vivo nanoparticle use is the characterization of biodistribution profiles. Such studies improve our understanding of particle uptake, specificity, and clearance mechanisms. Currently, the most prevalent nanoparticle biodistribution methods provide either aspatial quantification of whole-organ particle accumulation or nanometerresolution images of uptake in single cells. Few existing techniques are well-suited to study particle uptake on the micron to millimeter scales relevant to sub-tissue physiology. Here we demonstrate a new method called Hyperspectral Microscopy with Adaptive Detection (HSM-AD) that uses machine learning classification of hyperspectral dark-field images to study interactions between tissues and administered nanoparticles. This label-free, non-destructive method enables quantitative particle identification in histological sections and detailed observations of sub-organ accumulation patterns consistent with organ-specific clearance mechanisms, particle size, and the molecular specificity of the nanoparticle surface. Unlike studies with electron microscopy, HSM-AD is readily applied for large fields of view. HSM-AD achieves excellent detection sensitivity (99.4%) and specificity (99.7%) and can identify single nanoparticles. To demonstrate HSM-AD’s potential for novel nanoparticle uptake studies, we collected the first data on the sub-organ localization of large gold nanorods (LGNRs) in mice. We also observed differences in particle accumulation and localization patterns in tumors as a function of conjugated molecular targeting moieties. Thus, HSM-AD affords new degrees of detail for the study of nanoparticle uptake at physiological scales. HSM-AD may offer an auxiliary or alternative approach to study the biodistribution profiles of existing and novel nanoparticles.
In this study, we developed and applied highly-scattering large gold nanorods (LGNRs) and custom spectral detection
algorithms for high sensitivity contrast-enhanced optical coherence tomography (OCT). We were able to detect LGNRs
at a concentration as low as 50 pM in blood. We used this approach for noninvasive 3D imaging of blood vessels deep in
solid tumors in living mice. Additionally, we demonstrated multiplexed imaging of spectrally-distinct LGNRs that
enabled observations of functional drainage in lymphatic networks. This method, which we call MOZART, provides a
platform for molecular imaging and characterization of tissue noninvasively at cellular resolution.
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.
Nanoparticles can be synthesized in a wide array of shapes and sizes to suit specific biomedical applications in therapy and imaging. Prerequisite to such applications are particle stability in biological environments, non-toxicity, and facile conjugation of the particle surface with targeting biological moieties (such as antibodies). Here we report significant flaws in the common methods used to functionalize the surface of gold nanorods (GNRs) of larger-than-usual sizes. We find that while GNRs of sizes smaller than 50 x 15 nm can be effectively stabilized by polyethylene glycol (PEG)-based methods, larger GNRs form major aggregates and crash under similar functionalization conditions. Large GNRs may provide enhanced imaging sensitivity in biological applications due to greater optical extinction cross sections, provided that the GNRs can be made biostable. In this study, GNRs of sizes up to 90 x 30 nm were synthesized using two different published methods. Particle morphology and size distributions were characterized using Transmission Electron Microscopy (TEM), and optical spectra were measured by Vis-NIR Spectrometry. The colloidal stability of different-sized GNRs was assayed at various stages of functionalization using zeta potential and Vis-NIR measurements. The results of these experiments indicate that large GNRs functionalized with PEG undergo irreversible aggregation after minimal washing. We find that coating large GNRs with polystyrene sulfonate (PSS) instead of PEG vastly improves GNR stability in water and serum. Moreover, we provide a novel platform for conjugating biomolecules of interest to PSS-coated large GNRs. We show that larger GNRs produce stronger photoacoustic signal than commonly used smaller GNRs, indicating an advantage of using large GNRs for biomedical imaging. Our observations underscore that the biomedical advantages of novel nanoparticle synthesis methods may not be realized without tailored surface functionalization methods. More generally, our results suggest that materially-identical nanoparticles (i.e. GNRs) exhibit varying stability as a function of particle size.
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