Spectral- and Polarization-Dependent Scattering of Gold Nanobipyramids for Exogenous Contrast in Optical Coherence Tomography.

Polarization-sensitive optical coherence tomography (PS-OCT) reveals the subsurface microstructure of biological tissue and provides information regarding the polarization state of light backscattered from tissue. Complementing OCT's structural signal with molecular imaging requires strategies to simultaneously detect multiple exogenous contrast agents with high specificity in tissue. Specific detection of molecular probes enables the parallel visualization of physiological, cellular, and molecular processes. Here we demonstrate that, by combining PS-OCT and spectral contrast (SC)-OCT measurements, we can distinguish signatures of different gold nanobipyramids (GNBPs) in lymphatic vessels from the surrounding tissue and blood vessels in live mouse models. This technique could well be extended to other anisotropic nanoparticle-based OCT contrast agents and presents significant progress toward enabling OCT molecular imaging.

Gold Nanobipyramids as Second Near Infrared Optical Coherence Tomography Contrast Agents for in Vivo Multiplexing Studies.

Developing contrast-enhanced optical coherence tomography (OCT) techniques is important for specific imaging of tissue lesions, molecular imaging, cell-tracking, and highly sensitive microangiography and lymphangiography. Multiplexed OCT imaging in the second near-infrared (NIR-II) window is highly desirable since it allows simultaneous imaging and tracking of multiple biological events in high resolution with deeper tissue penetration in vivo. Here we demonstrate that gold nanobipyramids can function as OCT multiplexing contrast agents, allowing high-resolution imaging of two separate lymphatic flows occurring simultaneously from different drainage basins into the same lymph node in a live mouse. Contrast-enhanced multiplexed lymphangiography of a melanoma tumor in vivo shows that the peritumoral lymph flow upstream of the tumor is unidirectional, and tumor is accessible to such flow. Whereas the lymphatic drainage coming out from the tumor is multidirectional. We also demonstrate real-time tracking of the contrast agents draining from a melanoma tumor specifically to the sentinel lymph node of the tumor and the three-dimensional distribution of the contrast agents in the lymph node.

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.

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.

Optical coherence contrast imaging using gold nanorods in living mice eyes.

BACKGROUND: Optical coherence tomography (OCT) is a powerful imaging modality to visualize tissue structures, with axial image pixel resolution as high as 1.6 µm in tissue. However, OCT is intrinsically limited to providing structural information as the OCT contrast is produced by optically scattering tissues. METHODS: Gold nanorods (GNRs) were injected into the anterior chamber (AC) and cornea of mice eyes which could create a significant OCT signal and hence could be used as a contrast agent for in vivo OCT imaging. RESULTS: A dose of 30 nM of GNRs (13 nm in diameter and 45 nm in length) were injected to the AC of mice eyes and produced an OCT contrast nearly 50-fold higher than control mice injected with saline. Furthermore, the lowest detectable concentration of GNRs in living mice AC was experimentally estimated to be as low as 120 pM. CONCLUSIONS: The high sensitivity and low toxicity of GNRs brings great promise for OCT to uniquely become a high-resolution molecular imaging modality.

Noninvasive virtual biopsy using micro-registered optical coherence tomography (OCT) in human subjects.

Histological hematoxylin and eosin-stained (H&E) tissue sections are used as the gold standard for pathologic detection of cancer, tumor margin detection, and disease diagnosis. Producing H&E sections, however, is invasive and time-consuming. While deep learning has shown promise in virtual staining of unstained tissue slides, true virtual biopsy requires staining of images taken from intact tissue. In this work, we developed a micron-accuracy coregistration method [micro-registered optical coherence tomography (OCT)] that can take a two-dimensional (2D) H&E slide and find the exact corresponding section in a 3D OCT image taken from the original fresh tissue. We trained a conditional generative adversarial network using the paired dataset and showed high-fidelity conversion of noninvasive OCT images to virtually stained H&E slices in both 2D and 3D. Applying these trained neural networks to in vivo OCT images should enable physicians to readily incorporate OCT imaging into their clinical practice, reducing the number of unnecessary biopsy procedures.

Rapid Cellular-Resolution Skin Imaging with Optical Coherence Tomography Using All-Glass Multifocal Metasurfaces.

Cellular-resolution optical coherence tomography (OCT) is a powerful tool offering noninvasive histology-like imaging. However, like other optical microscopy tools, a high numerical aperture (N.A.) lens is required to generate a tight focus, generating a narrow depth of field, which necessitates dynamic focusing and limiting the imaging speed. To overcome this limitation, we developed a metasurface platform that generates multiple axial foci, which multiplies the volumetric OCT imaging speed by offering several focal planes. This platform offers accurate and flexible control over the number, positions, and intensities of axial foci generated. All-glass metasurface optical elements 8 mm in diameter are fabricated from fused-silica wafers and implemented into our scanning OCT system. With a constant lateral resolution of 1.1 µm over all depths, the multifocal OCT triples the volumetric acquisition speed for dermatological imaging, while still clearly revealing features of stratum corneum, epidermal cells, and dermal-epidermal junctions and offering morphological information as diagnostic criteria for basal cell carcinoma. The imaging speed can be further improved in a sparse sample, e.g., 7-fold with a seven-foci beam. In summary, this work demonstrates the concept of metasurface-based multifocal OCT for rapid virtual biopsy, further providing insights for developing rapid volumetric imaging systems with high resolution and compact volume.

Optical-resolution photoacoustic microscopy with a needle-shaped beam.

Optical-resolution photoacoustic microscopy (OR-PAM) can visualize wavelength-dependent optical absorption at the cellular level. However, OR-PAM suffers from a limited depth of field (DOF) due to the tight focus of the optical excitation beam, making it challenging to acquire high-resolution images of samples with uneven surfaces or high-quality volumetric images without z-scanning. To overcome this limitation, we propose needle-shaped beam photoacoustic microscopy (NB-PAM), which can extend the DOF to up to ~28-fold Rayleigh lengths via customized diffractive optical elements (DOEs). The DOE generate a needle beam with a well-maintained beam diameter, a uniform axial intensity distribution, and negligible sidelobes. The advantage of using NB-PAM is demonstrated by both histology-like imaging of fresh slide-free organs using a 266 nm laser and in vivo mouse brain vasculature imaging using a 532 nm laser. The approach provides new perspectives for slide-free intraoperative pathological imaging and in-vivo organ-level imaging.

Flexible method for generating needle-shaped beams and its application in optical coherence tomography.

Needle-shaped beams (NBs) featuring a long depth-of-focus (DOF) can drastically improve the resolution of microscopy systems. However, thus far, the implementation of a specific NB has been onerous due to the lack of a common, flexible generation method. Here we develop a spatially multiplexed phase pattern that creates many axially closely spaced foci as a universal platform for customizing various NBs, allowing flexible manipulations of beam length and diameter, uniform axial intensity, and sub-diffraction-limit beams. NBs designed via this method successfully extended the DOF of our optical coherence tomography (OCT) system. It revealed clear individual epidermal cells of the entire human epidermis, fine structures of human dermal-epidermal junction in a large depth range, and a high-resolution dynamic heartbeat of alive Drosophila larvae.

Intravital 3D visualization and segmentation of murine neural networks at micron resolution.

Optical coherence tomography (OCT) allows label-free, micron-scale 3D imaging of biological tissues' fine structures with significant depth and large field-of-view. Here we introduce a novel OCT-based neuroimaging setting, accompanied by a feature segmentation algorithm, which enables rapid, accurate, and high-resolution in vivo imaging of 700 µm depth across the mouse cortex. Using a commercial OCT device, we demonstrate 3D reconstruction of microarchitectural elements through a cortical column. Our system is sensitive to structural and cellular changes at micron-scale resolution in vivo, such as those from injury or disease. Therefore, it can serve as a tool to visualize and quantify spatiotemporal brain elasticity patterns. This highly transformative and versatile platform allows accurate investigation of brain cellular architectural changes by quantifying features such as brain cell bodies' density, volume, and average distance to the nearest cell. Hence, it may assist in longitudinal studies of microstructural tissue alteration in aging, injury, or disease in a living rodent brain.

Ultrahigh sensitivity carbon nanotube agents for photoacoustic molecular imaging in living mice.

Photoacoustic imaging is an emerging modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a novel photoacoustic contrast agent, Indocyanine Green dye-enhanced single walled carbon nanotube (SWNT-ICG). We conjugated this contrast agent with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the alpha(v)beta(3) integrins, which are associated with tumor angiogenesis. Intravenous administration of this tumor-targeted contrast agent to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. The new contrast agent gave a markedly 300 times higher photoacoustic contrast in living tissues than previously reported SWNTs, leading to subnanomolar sensitivities. Finally, we show that the new contrast agent can detect approximately 20 times fewer cancer cells than previously reported SWNTs.

Gold nanoparticles to enhance ophthalmic imaging.

The use of gold nanoparticles as diagnostic tools is burgeoning, especially in the cancer community with a focus on theranostic applications to both cancer diagnosis and treatment. Gold nanoparticles have also demonstrated great potential for use in diagnostic and therapeutic approaches in ophthalmology. Although many ophthalmic imaging modalities are available, there is still a considerable unmet need, in particular for ophthalmic molecular imaging for the early detection of eye disease before morphological changes are more grossly visible. An understanding of how gold nanoparticles are leveraged in other fields could inform new ways they could be utilized in ophthalmology. In this paper, we review current ophthalmic imaging techniques and then identify optical coherence tomography (OCT) and photoacoustic imaging (PAI) as the most promising technologies amenable to the use of gold nanoparticles for molecular imaging. Within this context, the development of gold nanoparticles as OCT and PAI contrast agents are reviewed, with the most recent developments described in detail.

Gold nanomaterials for optical biosensing and bioimaging.

Gold nanoparticles (AuNPs) are highly compelling nanomaterials for biomedical studies due to their unique optical properties. By leveraging the versatile optical properties of different gold nanostructures, the performance of biosensing and biomedical imaging can be dramatically improved in terms of their sensitivity, specificity, speed, contrast, resolution and penetration depth. Here we review recent advances of optical biosensing and bioimaging techniques based on three major optical properties of AuNPs: surface plasmon resonance, surface enhanced Raman scattering and luminescence. We summarize the fabrication methods and optical properties of different types of AuNPs, highlight the emerging applications of these AuNPs for novel optical biosensors and biomedical imaging innovations, and discuss the future trends of AuNP-based optical biosensors and bioimaging as well as the challenges of implementing these techniques in preclinical and clinical investigations.

Photoacoustic ocular imaging.

We developed a photoacoustic ocular imaging device and demonstrated its utility in imaging the deeper layers of the eye including the retina, choroid, and optic nerve. Using safe laser intensity, the photoacoustic system was able to visualize the blood distribution of an enucleated pig's eye and an eye of a living rabbit. Ultrasound images, which were simultaneously acquired, were overlaid on the photoacoustic images to visualize the eye's anatomy. Such a system may be used in the future for early detection and improved management of neovascular ocular diseases, including wet age-related macular degeneration and proliferative diabetic retinopathy.

Nanotherapeutic Shots through the Heart of Plaque.

The past several decades have brought significant advances in the application of clinical and preclinical nanoparticulate drugs in the field of cancer, but nanodrug development in cardiovascular disease has lagged in comparison. Improved understanding of the spatiotemporal kinetics of nanoparticle delivery to atherosclerotic plaques is required to optimize preclinical nanodrug delivery and to drive their clinical translation. Mechanistic studies using super-resolution and correlative light microscopy/electron microscopy permit a broad, ultra-high-resolution picture of how endothelial barrier integrity impacts the enhanced permeation and retention (EPR) effect for nanoparticles as a function of both atherosclerosis progression and metabolic therapy. Studies by Beldman et al. in the December issue of ACS Nano suggest atherosclerotic plaque progression supports endothelial junction stabilization, which can reduce nanoparticle entry into plaques, and metabolic therapy may induce similar effects. Herein, we examine the potential for advanced dynamic intravital microscopy-based mechanistic studies of nanoparticle entry into atherosclerotic plaques to shed light on the advantages of free extravasation versus immune-mediated nanoparticle uptake for effective clinical translation. We further explore the potential combination of metabolic therapy with another emerging cardiovascular disease treatment paradigm-efferocytosis stimulation-to enhance atherosclerotic plaque regression.

Optical Microscopy and Coherence Tomography of Cancer in Living Subjects.

Intravital microscopy (IVM) and optical coherency tomography (OCT) are two powerful optical imaging tools that allow visualization of dynamic biological activities in living subjects with subcellular resolutions. Recent advances in labeling and label-free techniques empower IVM and OCT for a wide range of preclinical and clinical cancer imaging, providing profound insights into the complex physiological, cellular, and molecular behaviors of tumors. Preclinical IVM and OCT have elucidated many otherwise inscrutable aspects of cancer biology, while clinical applications of IVM and OCT are revolutionizing cancer diagnosis and therapies. We review important progress in the fields of IVM and OCT for cancer imaging in living subjects, highlighting key technological developments and their emerging applications in fundamental cancer biology research and clinical oncology investigation.

Difference-Frequency Ultrasound Imaging With Non-Linear Contrast.

Conventional ultrasound imaging is based on the scattering of sound from inhomogeneities in the density and the speed of sound and is often used in medicine to resolve pathologic compared to normal tissue. Here we demonstrate a difference-frequency ultrasound (dfUS) imaging method that is based on the interaction of two sound pulses that propagate non-collinearly and intersect in space and time. The dfUS signal arises primarily from the second-order non-linear coefficient, a contrast mechanism that differs from linear and harmonic US imaging. The distinct contrast mechanism allows dfUS to image anatomic features that are not identifiable in conventional US images of salmon and pig kidney tissue. Further, dfUS produces enhanced contrast of glioblastoma tumor implanted in the mouse brain, revealing its potential for improving medical diagnosis. Progress towards a real-time system is discussed.

Angular compounding for speckle reduction in optical coherence tomography using geometric image registration algorithm and digital focusing.

Optical coherence tomography (OCT) suffers from speckle noise due to the high spatial coherence of the utilized light source, leading to significant reductions in image quality and diagnostic capabilities. In the past, angular compounding techniques have been applied to suppress speckle noise. However, existing image registration methods usually guarantee pure angular compounding only within a relatively small field of view in the focal region, but produce spatial averaging in the other regions, resulting in resolution loss and image blur. This work develops an image registration model to correctly localize the real-space location of every pixel in an OCT image, for all depths. The registered images captured at different angles are fused into a speckle-reduced composite image. Digital focusing, based on the convolution of the complex OCT images and the conjugate of the point spread function (PSF), is studied to further enhance lateral resolution and contrast. As demonstrated by experiments, angular compounding with our improved image registration techniques and digital focusing, can effectively suppress speckle noise, enhance resolution and contrast, and reveal fine structures in ex-vivo imaged tissue.

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.

Upper limit for angular compounding speckle reduction.

Angular compounding is a technique for reducing speckle noise in optical coherence tomography that is claimed to significantly improve the signal-to-noise ratio (SNR) of images without impairing their spatial resolution. Here, we examine how focal point movements caused by optical aberrations in an angular compounding system may produce unintended spatial averaging and concomitant loss of spatial resolution. Experimentally, we accounted for such aberrations by aligning our system and measuring distortions in images and found that when the distortions were corrected, the speckle reduction by angular compounding was limited. Our theoretical analysis using Monte Carlo simulations indicates that "pure" angular compounding (i.e., with no spatial averaging) over our full numerical aperture (13° in air) can improve the SNR by not more than a factor of 1.3. Illuminating only a partial aperture cannot improve this factor compared to a spatial averaging system with equivalent loss of resolution. We conclude that speckle reduction using angular compounding is equivalent to spatial averaging. Nonetheless, angular compounding may be useful for improving images in applications where the depth of field is important. The distortions tend to be the greatest off the focal plane, and so angular compounding combined with our correction technique can reduce speckle with a minimal loss of resolution across a large depth of field.