Three-dimensional sub-100 nm super-resolution imaging of biological samples using a phase ramp in the objective pupil

Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional (3D) localisation microscopy by engineering of the point-spread function (PSF) of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions (standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively). The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.      

Super‐Resolution Nonlinear Photothermal Microscopy

Super‐resolution fluorescence microscopy enables imaging of fluorescent structures beyond the diffraction limit. However, this technique cannot be applied to weakly fluorescent cellular components or labels. As an alternative, photothermal microscopy based on nonradiative transformation of absorbed energy into heat has demonstrated imaging of nonfluorescent structures including single molecules and ~1‐nm gold nanoparticles. However, previously photothermal imaging has been performed with a diffraction‐limited resolution only. Herein, super‐resolution, far‐field photothermal microscopy based on nonlinear signal dependence on the laser energy is introduced. Among various nonlinear phenomena, including absorption saturation, multiphoton absorption, and signal temperature dependence, signal amplification by laser‐induced nanobubbles around overheated nano‐objects is explored. A Gaussian laser beam profile is used to demonstrate the image spatial sharpening for calibrated 260‐nm metal strips, resolving of a plasmonic nanoassembly, visualization of 10‐nm gold nanoparticles in graphene, and hemoglobin nanoclusters in live erythrocytes with resolution down to 50 nm. These nonlinear phenomena can be used for 3D imaging with improved lateral and axial resolution in most photothermal methods, including photoacoustic microscopy.     

High‐Resolution 3D NIR‐II Photoacoustic Imaging of Cerebral and Tumor Vasculatures Using Conjugated Polymer Nanoparticles as Contrast Agent

Exogenous contrast‐agent‐assisted NIR‐II optical‐resolution photoacoustic microscopy imaging (ORPAMI) holds promise to decipher wide‐field 3D biological structures with deep penetration, large signal‐to‐background ratio (SBR), and high maximum imaging depth to depth resolution ratio. Herein, NIR‐II conjugated polymer nanoparticle (CP NP) assisted ORPAMI is reported for pinpointing cerebral and tumor vasculatures. The CP NPs exhibit a large extinction coefficient of 48.1 L g^?1 at the absorption maximum of 1161 nm, with an ultrahigh PA sensitivity up to 2 µg mL^?1. 3D ORPAMI of wide‐field mice ear allows clear visualization of regular vasculatures with a resolution of 19.2 µm and an SBR of 29.3 dB at the maximal imaging depth of 539 µm. The margin of ear tumor composed of torsional dense vessels among surrounding normal regular vessels can be clearly delineated via 3D angiography. In addition, 3D whole‐cortex cerebral vasculatures with large imaging area (48 mm²), good resolution (25.4 µm), and high SBR (22.3 dB) at a depth up to 1001 µm are clearly resolved through the intact skull. These results are superior to the recently reported 3D NIR‐II fluorescence confocal vascular imaging, which opens up new opportunities for NIR‐II CP‐NP‐assisted ORPAMI in various biomedical applications.     

Focused Orientation and Position Imaging (FOPI) of Single Anisotropic Plasmonic Nanoparticles by Total Internal Reflection Scattering Microscopy

The defocused orientation and position imaging (DOPI) and polarization-based in-focus imaging techniques have been widely used for detecting rotational motions with anisotropic gold nanorods (AuNRs) as orientation probes. However, these techniques have a number of significant limitations, such as the greatly reduced signal intensity and relatively low spatial and temporal resolutions for out-of-focus AuNRs and the angular degeneracy for in-focus AuNRs. Herein, we present a total internal reflection (TIR) scattering-based focused orientation and position imaging (FOPI) of AuNRs supported on a 50 nm thick gold film, which enables us to overcome the aforementioned limitations. Imaging AuNRs under the TIR scattering microscope provides excellent signal-to-noise ratio and results in no deteriorating images. The scattering patterns of AuNRs on the gold substrate are affected by the strong interaction of the excited dipole in the AuNR with the image dipole in the gold substrate. The doughnut-shaped scattering field distribution allows for high-throughput determination of the three-dimensional spatial orientation of in-focus AuNRs within a single frame without angular degeneracy. Therefore, the TIR scattering-based FOPI method is demonstrated to be an outstanding candidate for studying dynamics of functionalized nanoparticles on a large variety of functional surfaces.     

Aggregation‐Induced Nonlinear Optical Effects of AIEgen Nanocrystals for Ultradeep In Vivo Bioimaging

Nonlinear optical microscopy has become a powerful tool in bioimaging research due to its unique capabilities of deep optical sectioning, high‐spatial‐resolution imaging, and 3D reconstruction of biological specimens. Developing organic fluorescent probes with strong nonlinear optical effects, in particular third‐harmonic generation (THG), is promising for exploiting nonlinear microscopic imaging for biomedical applications. Herein, a simple method for preparing organic nanocrystals based on an aggregation‐induced emission (AIE) luminogen (DCCN) with bright near‐infrared emission is successfully demonstrated. Aggregation‐induced nonlinear optical effects, including two‐photon fluorescence (2PF), three‐photon fluorescence (3PF), and THG, of DCCN are observed in nanoparticles, especially for crystalline nanoparticles. The nanocrystals of DCCN are successfully applied for 2PF microscopy at 1040 nm NIR‐II excitation and THG microscopy at 1560 nm NIR‐II excitation, respectively, to reconstruct the 3D vasculature of the mouse cerebral vasculature. Impressively, the THG microscopy provides much higher spatial resolution and brightness than the 2PF microscopy and can visualize small vessels with diameters of ≈2.7 µm at the deepest depth of 800 µm in a mouse brain. Thus, this is expected to inspire new insights into the development of advanced AIE materials with multiple nonlinearity, in particular THG, for multimodal nonlinear optical microscopy.     

Silica shell-assisted synthetic route for mono-disperse persistent nanophosphors with enhanced in vivo recharged near-infrared persistent luminescence

Near-infrared (NIR) persistent-luminescence nanoparticles have emerged as a new class of background-free contrast agents that are promising for in vivo imaging.The next key roadblock is to establish a robust and controllable method for synthesizing monodisperse nanoparticles with high luminescence brightness and long persistent duration.Herein,we report a synthesis strategy involving the coating/etching of the SiO2 shell to obtain a new class of small NIR highly persistent luminescent ZnGa2O4∶Cr3+,Sn4+ (ZGOCS) nanoparticles.The optimized ZGOCS nanoparticles have an excellent size distribution of ～15 nm without any agglomeration and an NIR persistent luminescence that is enhanced by a factor of 13.5,owing to the key role of the SiO2 shell in preventing nanoparticle agglomeration after annealing.The ZGOCS nanoparticles have a signal-to-noise ratio ～3 times higher than that of previously reported ZnGa2O4∶Cr3+ (ZGC-1) nanoparticles as an NIR persistent-luminescence probe for in vivo bioimaging.Moreover,the persistent-luminescence signal from the ZGOCS nanoparticles can be repeatedly re-charged in situ with external excitation by a white lightemitting diode;thus,the nanoparticles are suitable for long-term in vivo imaging applications.Our study suggests an improved strategy for fabricating novel high-performance optical nanoparticles with good biocompatibility.     

一种用于微米／纳米硬度测试的具有纳米分辨率的压痕成像系统

与包括原子力显微镜在内的其他压痕形貌测量方法相比,光学显微镜以其非接触、大测量范围及较快的测量速度等优点在硬度测试中应用广泛,但包括共焦显微镜在内的常规显微镜其分辨率,特别是纵向分辨率,一直不能满足要求．为此开发了一种沿光轴方向的具有纳米分辨率的压痕成像系统．该系统基于层析全场显微术,即在常规光学显微镜的基础上引入结构光照明．应用移相方法得到样品的三维形貌．成像系统样机的测量结果表明,系统的纵向分辨率可达2nm,可以高精度地实现压痕的三维形貌测量,并量化揭示纳米压痕测试中常见的“坟起（pile-up）”及“沉入（sink-in）”现象．该系统有助于进一步研究显微及纳米硬度计量方法并降低计量的不确定度.     

Three‐Dimensional Exploration and Mechano‐Biophysical Analysis of the Inner Structure of Living Cells

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self‐interference digital holographic microscopy. This biophotonic holographic workstation enables non‐contact, minimally invasive, flexible, high‐precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 μm diameter are three‐dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.     

Wide-Field Three-Dimensional Depth-Invariant Cellular-Resolution Imaging of the Human Retina

Three-dimensional (3D) cellular-resolution imaging of the living human retina over a large field of view will bring a great impact in clinical ophthalmology, potentially finding new biomarkers for early diagnosis and improving the pathophysiological understanding of ocular diseases. While hardware-based and computational adaptive optics (AO) optical coherence tomography (OCT) have been developed to achieve cellular-resolution retinal imaging, these approaches support limited 3D imaging fields, and their high cost and intrinsic hardware complexity limit their practical utility. Here, this work demonstrates 3D depth-invariant cellular-resolution imaging of the living human retina over a 3 × 3 mm field of view using the first intrinsically phase-stable multi-MHz retinal swept-source OCT and novel computational defocus and aberration correction methods. Single-acquisition imaging of photoreceptor cells, retinal nerve fiber layer, and retinal capillaries is presented across unprecedented imaging fields. By providing wide-field 3D cellular-resolution imaging in the human retina using a standard point-scan architecture routinely used in the clinic, this platform proposes a strategy for expanded utilization of high-resolution retinal imaging in both research and clinical settings.     

Single Molecule Localization Microscopy for Studying Small Extracellular Vesicles

Small extracellular vesicles (sEVs) are 30–200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200–300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.     

X-ray orientation microscopy using topo-tomography and multi-mode diffraction contrast tomography

Polycrystal orientation mapping techniques based on full-field acquisition schemes like X-ray Diffraction Contrast Tomography and certain other variants of 3D X-ray Diffraction or near-field High Energy Diffraction Microscopy enable time efficient mapping of 3D grain microstructures. The spatial resolution obtained with this class of monochromatic beam X-ray diffraction imaging approaches remains typically below the ultimate spatial resolution achievable with X-ray imaging detectors. Introducing a generalised reconstruction framework enabling the combination of acquisitions with different detector pixel size and sample tilt settings provide a pathway towards 3D orientation mapping with a spatial resolution approaching the one of state of the art X-ray imaging detector systems.     

Dual‐Modality Surface‐Enhanced Resonance Raman Scattering and Multispectral Optoacoustic Tomography Nanoparticle Approach for Brain Tumor Delineation

Difficulty in visualizing glioma margins intraoperatively remains a major issue in the achievement of gross total tumor resection and, thus, better clinical outcome of glioblastoma (GBM) patients. Here, the potential of a new combined optical + optoacoustic imaging method for intraoperative brain tumor delineation is investigated. A strategy using a newly developed gold nanostar synthesis method, Raman reporter chemistry, and silication method to produce dual‐modality contrast agents for combined surface‐enhanced resonance Raman scattering (SERRS) and multispectral optoacoustic tomography (MSOT) imaging is devised. Following intravenous injection of the SERRS‐MSOT‐nanostars in brain tumor bearing mice, sequential MSOT imaging is performed in vivo and followed by Raman imaging. MSOT is able to accurately depict GBMs three‐dimensionally with high specificity. The MSOT signal is found to correlate well with the SERRS images. Because SERRS enables uniquely sensitive high‐resolution surface detection, it could represent an ideal complementary imaging modality to MSOT, which enables real‐time, deep tissue imaging in 3D. This dual‐modality SERRS‐MSOT‐nanostar contrast agent reported here is shown to enable high precision depiction of the extent of infiltrating GBMs by Raman‐ and MSOT imaging in a clinically relevant murine GBM model and could pave new ways for improved image‐guided resection of brain tumors.     

A hybrid numerical and imaging approach for characterizing defects in composite structures

In this study, a hybrid approach coupling hyperspectral near infrared imaging with a progressive finite element method is proposed for characterization of the elastic and failure response of composites with non-uniform variations of the wrinkles profile through the thickness and across the structure dimensions. In this approach, hyperspectral near infrared spectroscopy is used to create a 3D profile of the surface resin pockets with the capability of measuring resin thickness from approximately 125 to 2500 μm. These resin pockets are directly correlated to underlying ply level wrinkling as confirmed by optical microscopy. The 3D mapped resin plane obtained from the hyperspectral imaging is used to morph a ply-by-ply finite element model of a carbon-fiber/epoxy resin laminated plate using a progressive damage failure methodology. The results show the capability of the hybrid method to predict the structural response in laminated composites containing spatially distributed and non-uniform ply-level wrinkling.     

Nanoparticles for Optical Molecular Imaging of Atherosclerosis

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope‐compatible optical imaging techniques would offer a stand‐alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high‐quality optical‐contrast‐enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

     

Smart‐Dust‐Nanorice for Enhancement of Endogenous Raman Signal,Contrast in Photoacoustic Imaging,and T2‐Shortening in Magnetic Resonance Imaging

Raman microspectroscopy provides chemo‐selective image contrast, sub‐micrometer resolution, and multiplexing capabilities. However, it suffers from weak signals resulting in image‐acquisition times of up to several hours. Surface‐enhanced Raman scattering (SERS) can dramatically enhance signals of molecules in close vicinity of metallic surfaces and overcome this limitation. Multimodal, SERS‐active nanoparticles are usually labeled with Raman marker molecules, limiting SERS to the coating material. In order to realize multimodal imaging while acquiring the rich endogenous vibronic information of the specimen, a core–shell particle based on “Nanorice”, where a spindle‐shaped iron oxide core is encapsulated by a closed gold shell, is developed. An ultrathin layer of silica prevents agglomeration and unwanted chemical interaction with the specimen. This approach provides Raman signal enhancement due to plasmon resonance effects of the shell while the optical absorption in the near‐infrared spectral region provides contrast in photoacoustic tomography. Finally, T2‐relaxation of a magnetic resonance imaging (MRI) experiment is altered by taking advantage of the iron oxide core. The feasibility for Raman imaging is evaluated by nearfield simulations and experimental studies on the primate cell line COS1. MRI and photoacoustics are demonstrated in agarose phantoms illustrating the promising translational nature of this strategy for clinical applications in radiology.     

In Vivo Volumetric Photoacoustic Molecular Angiography and Therapeutic Monitoring with Targeted Plasmonic Nanostars

Photoacoustic (PA) imaging promises deeper tissue penetration while maintaining rich optical contrast as compared to other high resolution optical imaging techniques. In this report, a near‐infrared pulse laser serves as the excitation source, and 128 ultrasonic transducers are spirally distributed on a hemispherical surface to receive PA signals for three‐dimensional (3D) image reconstruction. With these attributes, the unique modality produces an isotropic and homogeneous spatial resolution (～200 μm) with penetration depth of centimeters. Cyclic Arg‐Gly‐Asp (RGD) peptides conjugated plasmonic gold nanostars (RGD‐GNS) are designed to specifically target over‐expressed integrin α_vβ₃ on tumor neovasculature, enabling highly sensitive angiography and photothermal therapy (PTT). After the administration of RGD‐GNS, tumor angiogenesis is clearly imaged with enhanced contrast, and the growth of tumor is effectively inhibited by PTT after laser irradiation. This study suggest that the PA angiography with plasmonic RGD‐GNS can be applied as a triple functional platform for tumor diagnosis, PTT, and treatment monitoring. This PA technique offers deeper imaging depth with homogeneous resolution over existing optical imaging techniques for early diagnosis of tumor angiogenesis as well as on‐the‐spot nanotherapeutic evaluation.     

Three-dimensional optical tomography: resolution in small-object imaging

Near-infrared (NIR) optical tomography provide estimates of the internal distribution of optical absorption and transport scattering from boundary measurement of light propagation within biological tissue. Although this is a truly three-dimensional (3D) imaging problem, most research to date has concentrated on two-dimensional modeling and image reconstruction. More recently, 3D imaging algorithms are demonstrating better estimation of the light propagation within the imaging region and are providing the basis of more accurate image construction algorithms. As 3D methods emerge, it will become increasingly important to evaluate their resolution, contrast, and localization of optical property heterogeneity. We present a concise study of 3D reconstructed resolution of a small, low-contrast, absorbing and scattering anomaly as it is placed in different locations within a cylindrical phantom. The object is an 8-mm-diameter cylinder, which represents a typical small target that needs to be resolved in NIR mammographic imaging. The best resolution and contrast is observed when the object is located near the periphery of the imaging region (12-22 mm from the edge) and is also positioned within the multiple measurement planes, with the most accurate results seen for the scatter image when the anomaly is at 17 mm from the edge. Furthermore, the accuracy of quantitative imaging is increased to almost 100% of the target values when a priori information regarding the internal structure of imaging domain is utilized.     

Comparison of imaging quality between linear sampling method and time reversal in microwave imaging problems

This research investigated imaging quality of two important methods widely used in electromagnetic inverse scattering problems. The algorithms, time reversal (TR) and linear sampling method (LSM), were compared for resolution of point imaging and a correlation indicator to determine image quality. Comparisons were made in single- and multifrequency modes for 2D scenarios in free-space. Comparisons revealed that resolution of TR is much better than LSM. In order to compare the total reconstructed images, several cases were considered to determine a comprehensive conclusion. The simulations were done based on experimental data. In this case, comparisons showed that in the term of correlation indicator, LSM surpasses TR.     

The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision

The double-helix point spread function microscope encodes the axial (z) position information of single emitters in wide-field (x,y) images, thus enabling localization in three dimensions (3D) inside extended volumes. We experimentally determine the statistical localization precision σ of this approach using single emitters in a cell under typical background conditions, demonstrating σ?相似文献   

Hybrid method combining orthogonal projection Fourier transform profilometry and laser speckle imaging for 3D visualization of flow profile

This paper reports a straightforward technique for three-dimensional (3D) visualization of a flow profile by a hybrid algorithm combining Fourier transform orthogonal fringe projection and laser speckle imaging techniques. The use of orthogonal projection aims to suppress the zero order allowing surface reconstruction with high spatial resolution and accuracy while analyzing the intensity fluctuations of diffuse backscattered laser light providing 2D flow information. Once both are achieved, 3D flow visualization can be displayed. The method is experimentally validated first with a plastic tube filled with scattering liquid (milk) running at various controlled flow rates and then with the tube embedded under scattering layers (chicken breast) of varying thickness. The system includes a single, common camera, a commercial projector (profilometry channel), a laser light source (flow channel), and a computer station. In addition, orthogonal projection processing was combined with Hilbert transform, increasing the visualization and resolution of the measured flow profile.