Micrometer Resolution Imaging Research Articles

Non-Optical, Label-free Electrical Capacitance Imaging of Microorganisms.

Microbes live in diverse environments, and occupy biological roles across many timescales. Investigating the full scope of microbial activity requires imaging systems appropriate to each context. Though optical microscopy is powerful, the use of light, lenses, and other hardware limits where it can be applied. At the same time, existing non-optical imaging methods are frequently destructive to samples and require extensive equipment. In this paper we present a non-optical imaging system that is small, cheap, requires no sample labeling, and is compatible with a variety of microbial species. Our system uses semiconductor chips to measure the inherent material properties of a sample with spatial sensitivity, producing images of microbes contrasted against their environment and each other. Our technique captures label-free, micrometer-resolution images with a pocket-sized device, enabling microbiological imaging experiments in new environments with new species.

Optical Coherence Tomography-Based Atlas of the Human Cochlear Hook Region.

Advancements in intracochlear diagnostics, as well as prosthetic and regenerative inner ear therapies, rely on a good understanding of cochlear microanatomy. The human cochlea is very small and deeply embedded within the densest skull bone, making nondestructive visualization of its internal microstructures extremely challenging. Current imaging techniques used in clinical practice, such as MRI and CT, fall short in their resolution to visualize important intracochlear landmarks, and histological analysis of the cochlea cannot be performed on living patients without compromising their hearing. Recently, optical coherence tomography (OCT) has been shown to be a promising tool for nondestructive micrometer resolution imaging of the mammalian inner ear. Various studies performed on human cadaveric tissue and living animals demonstrated the ability of OCT to visualize important cochlear microstructures (scalae, organ of Corti, spiral ligament, and osseous spiral lamina) at micrometer resolution. However, the interpretation of human intracochlear OCT images is non-trivial for researchers and clinicians who are not yet familiar with this novel technology. In this study, we present an atlas of intracochlear OCT images, which were acquired in a series of 7 fresh and 10 fresh-frozen human cadaveric cochleae through the round window membrane and describe the qualitative characteristics of visualized intracochlear structures. Likewise, we describe several intracochlear abnormalities, which could be detected with OCT and are relevant for clinical practice.

Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes

BackgroundMicrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and embedding. The exchange of solvents for the creation of sectionable paraffin blocks modifies tissue density and generates non-uniform brain shrinkage. New methodWe employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to paraffin embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology. ResultsThe volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements. Comparison with existing methodsMicrotomography provided sub-10 μm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation. ConclusionWe present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and embedding.

Imaging Motion: A Comprehensive Review of Optical Coherence Tomography Angiography.

Optical coherence tomography (OCT) is a three-dimensional (3-D) optical imaging technology that provides noninvasive, micrometer resolution images of structural interiors within biological samples with an approximately 1~2 mm penetration depth. Over the last decades, advances in OCT have revolutionized biomedical imaging by demonstrating a potential of optical biopsy in preclinical and clinical settings. Recently, functional OCT imaging has shown a promise as angiography to visualize cell-perfused vasculatures in the tissue bed in vivo without requiring any exogenous contrast agents. This new technology termed OCT angiography (OCTA) possesses a unique imaging capability of delineating tissue morphology and blood or lymphatic vessels down to capillaries at real-time acquisition rates. For the past 10years since 2007, OCTA has been proven to be a useful tool to identify disorder or dysfunction in tissue microcirculation from both experimental animal studies and clinical studies in ophthalmology and dermatology. In this section, we overview about OCTA including a basic principle of OCTA explained with simple optical physics, and its scan protocols and post-processing algorithms for acquisition of angiography. Then, potential and challenge of OCTA for clinical settings are shown with outcomes of human studies.

Long-Range Optical Coherence Tomography of the Neonatal Upper Airway for Early Diagnosis of Intubation-related Subglottic Injury.

Subglottic edema and acquired subglottic stenosis are potentially airway-compromising sequelae in neonates following endotracheal intubation. At present, no imaging modality is capable of in vivo diagnosis of subepithelial airway wall pathology as signs of intubation-related injury. To use Fourier domain long-range optical coherence tomography (LR-OCT) to acquire micrometer-resolution images of the airway wall of intubated neonates in a neonatal intensive care unit setting and to analyze images for histopathology and airway wall thickness. LR-OCT of the neonatal laryngotracheal airway was performed a total of 94 times on 72 subjects (age, 1-175 d; total intubation, 1-104 d). LR-OCT images of the airway wall were analyzed in MATLAB. Medical records were reviewed retrospectively for extubation outcome. Backward stepwise regression analysis demonstrated a statistically significant association between log(duration of intubation) and both laryngeal (P < 0.001; multiple r(2) = 0.44) and subglottic (P < 0.001; multiple r(2) = 0.55) airway wall thickness. Subjects with positive histopathology on LR-OCT images had a higher likelihood of extubation failure (odds ratio, 5.9; P = 0.007). Longer intubation time was found to be significantly associated with extubation failure. LR-OCT allows for high-resolution evaluation and measurement of the airway wall in intubated neonates. Our data demonstrate a positive correlation between laryngeal and subglottic wall thickness and duration of intubation, suggestive of progressive soft tissue injury. LR-OCT may ultimately aid in the early diagnosis of postintubation subglottic injury and help reduce the incidences of failed extubation caused by subglottic edema or acquired subglottic stenosis in neonates. Clinical trial registered with www.clinicaltrials.gov (NCT 00544427).

High-axial-resolution, full-field optical coherence microscopy using tungsten halogen lamp and liquid-crystal-based achromatic phase shifter.

A high-axial-resolution, full-field optical coherence microscope (FFOCM) for topography and tomography applications is presented. The FFOCM is based on a polarization Linnik interference microscope equipped with a tungsten halogen lamp. The phase difference between the reference and test beams in the microscope is precisely and quickly shifted by using an achromatic liquid-crystal phase shifter (LCPS). The cross-sectional amplitude and phase maps of an interferogram are retrieved by using a three-step phase-shifting technique. The LCPS consists of three identical nematic liquid-crystal (NLC) cells sandwiched between two quarter-wave plates so that it functions as a typical quarter-half-quarter phase shifter. Instead of using high-cost NLC cells with precise thickness of half-wave retardation, a method is proposed to operate thicker NLC cells without scarifying the axial resolution. Experimental results reveal that the FFOCM is able to perform three-dimensional micrometer-resolution imaging.

New High Stopping Power Thin Scintillators Based on ${\rm Lu}_{2}{\rm O}_{3}$ and ${\rm Lu}_{3}{\rm Ga}_{5-{\rm x}}{\rm In}_{\rm x}{\rm O}_{12}$ for High Resolution X-ray Imaging

X-ray computed tomography devices and X-ray diffraction techniques are powerful tools: the former provide volumetric data of samples during a non-destructive examination for biology and material science, and the latter measure grain orientation and strain, as well as crystalline phase identification and structure refinement. Today, the European Synchrotron Radiation Facility (ESRF) provides increasingly higher energy beams, up to 150 keV combined with higher brilliance (1013 X-ray photons/sec). This means that detectors suffer from low X-ray absorption at high spatial resolution (1-10 μm) and from radiation damage in tomography and diffraction applications. In addition, more and more experiments in medicine require the absorbed dose by the sample to be reduced. In this context, more efficient scintillators are developed and evaluated at the ESRF. In order to perform sub-micrometer and micrometer resolution imaging scintillators 1 μm to 500 μm thin are required. Single Crystal Film scintillators (SCF), 1 μm to 100 μm can be obtained via Liquid Phase Epitaxy for sub-micrometer resolution. Transparent ceramics, 100 μm to 500 μm thick are promising candidates for X-ray imaging requiring high X-ray absorption and good contrast with micrometer resolution. Commonly available scintillators, such as CdWO4 and YAG:Ce suffer from low efficiency, therefore new scintillators with higher light yield and stopping-power are required. A first test was carried out to evaluate an Europium doped Lutetium Oxide ceramic for micrometer resolution and new SCFs of Lu3Ga5-xInxO12:Eu for sub-micrometer resolution are investigated. Performance of Lu2O3 and LuInGG, i.e absorption, light yield, afterglow, spatial resolution will be presented and compared to standard screens (YAG, GGG). First results will be illustrated with X-ray images and will demonstrate the absorption efficiency improvement at high spatial resolution.

Bioimaging of trace metals in ancient Chilean mummies and contemporary Egyptian teeth by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS)

Distributional analysis of trace metals in biological tissue provides important archived toxicological and nutritional information. In this study, lead, strontium, zinc, and lithium concentrations were measured in dental tissue using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Teeth from two populations were sampled: ancient Chilean mummies (n=16) from Arica city as well as recently exfoliated deciduous teeth from contemporary Egyptian children (n=20) living in the Nile Delta. In this study, LA-ICP-MS was used to obtain high-resolution (50μm) elemental bioimages in dental tissue. The experiments consisted of fully quantitative Pb bioimages as well as semi-quantitative Zn and Li bioimages of one contemporary Nile Delta, Egypt tooth and one ancient tooth from the Andes. Lead levels in the Egyptian teeth averaged an order of magnitude greater than in the ancient Chilean teeth (14.93±32.28μg/g and 1.33±0.84μg/g, respectively), and had a much greater range of variation among teeth (1.02μg/g–68.38μg/g versus 0.36μg/g–2.35μg/g, respectively). Lithium, an element widely present in northern Chile, was found in higher concentrations in the Arica teeth than in the Kalama teeth, with the highest concentrations at two sites in Chilean burial grounds where water is tainted with natural lithium. Micrometer resolution images of quantified Pb, Zn, and Li concentrations from two teeth revealed higher concentrations of Pb and Zn in the pulp and surface enamel. Zn and Li are concentrated in the crown, potentially indicative of higher prenatal exposure.

Effects of mineral distribution at mesoscopic scale on solute diffusion in a clay‐rich rock: Example of the Callovo‐Oxfordian mudstone (Bure, France)

The mesostructure (millimeter to micrometer scale) of clay‐rich sedimentary rocks is generally characterized by a connected fine‐grained clay matrix embedding coarser nonclay minerals. We use the Callovo‐Oxfordian clay‐rich rock formation (France) to illustrate how mesostructure influences solute transfer in clay‐rich rocks at larger scales. Using micrometer resolution imaging techniques (SEM and micro‐CT) major mineral phases (clay matrix, carbonates, tectosilicates, and heavy minerals) were mapped both in two dimensional (2‐D) and three dimensional (3‐D) at the mesoscale. Orientation and elongation distributions of carbonate and tectosilicate grains measured on mineral maps reveal an anisotropic mesostructure relative to the bedding plane, in agreement with the geological history of the sedimentary rock. Diffusion simulations were performed based on the 3‐D mineral maps using a random walk method thus allowing direct computation of mesoscopic scale‐related diffusion anisotropy and tortuosity. Considering an isotropic clay matrix, simulated diffusion anisotropy (1.11–1.26) was found lower than the one experimentally measured on macroscopic samples (1.5 to 2), due to the anisotropy feature of pores within the clay matrix. The effects of the mineral content variations on diffusion properties were then investigated by numerical modifications of a mineral map combined with diffusion simulations. Evolution of the tortuosity and diffusion anisotropy with the clay matrix content were successfully interpreted by the Koponen percolation/diffusion model, whereas the Archie approach fails to reproduce diffusion properties at low clay contents. A comparison of fitting parameters with those obtained experimentally indicates that diffusion coefficient variations observed at a large scale could be mainly controlled by the mesostructure.

X‐Space MPI: Magnetic Nanoparticles for Safe Medical Imaging

One quarter of all iodinated contrast X-ray clinical imaging studies are now performed on Chronic Kidney Disease (CKD) patients. Unfortunately, the iodine contrast agent used in X-ray is often toxic to CKD patients' weak kidneys, leading to significant morbidity and mortality. Hence, we are pioneering a new medical imaging method, called Magnetic Particle Imaging (MPI), to replace X-ray and CT iodinated angiography, especially for CKD patients. MPI uses magnetic nanoparticle contrast agents that are much safer than iodine for CKD patients. MPI already offers superb contrast and extraordinary sensitivity. The iron oxide nanoparticle tracers required for MPI are also used in MRI, and some are already approved for human use, but the contrast agents are far more effective at illuminating blood vessels when used in the MPI modality. We have recently developed a systems theoretic framework for MPI called x-space MPI, which has already dramatically improved the speed and robustness of MPI image reconstruction. X-space MPI has allowed us to optimize the hardware for fi ve MPI scanners. Moreover, x-space MPI provides a powerful framework for optimizing the size and magnetic properties of the iron oxide nanoparticle tracers used in MPI. Currently MPI nanoparticles have diameters in the 10-20 nanometer range, enabling millimeter-scale resolution in small animals. X-space MPI theory predicts that larger nanoparticles could enable up to 250 micrometer resolution imaging, which would represent a major breakthrough in safe imaging for CKD patients.

In Vivo Imaging of Myelin in the Vertebrate Central Nervous System Using Third Harmonic Generation Microscopy

Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy—a coherent, nonlinear, dye-free imaging modality—provides micrometer resolution imaging of myelin in the mouse CNS. In fixed tissue, we found that THG signals arose from white matter tracts and were colocalized with two-photon excited fluorescence (2PEF) from a myelin-specific dye. In vivo, we used simultaneous THG and 2PEF imaging of the mouse spinal cord to resolve myelin sheaths surrounding individual fluorescently-labeled axons, and followed myelin disruption after spinal cord injury. Finally, we suggest optical mechanisms that underlie the myelin specificity of THG. These results establish THG microscopy as an ideal tool for the study of myelin loss and recovery.

Wettability characterization method based on optical coherence tomography imaging

A novel method for surface wetting characterization based on contact angle measurements is presented. Three dimensional structural imaging of a droplet on a substrate is performed using optical coherence tomography (OCT) which provides micrometer resolution images without contact with the sample. An automatic OCT intensity variation based, layer segmentation method was implemented to identify air-droplet, droplet-substrate and air-substrate interfaces. A glycerol droplet on a rough tilted glass substrate was used as a test sample to demonstrate the applicability of the proposed method for wetting characterization. Results show that the contact angle of any observation angle can be obtained. In addition, the droplet's average refractive index can be determined. The proposed method is an interesting and complementary tool for present wetting characterization methods.

PHASE DETECTION WITH SUB-NANOMETER SENSITIVITY USING POLARIZATION QUADRATURE ENCODING METHOD IN OPTICAL COHERENCE TOMOGRAPHY

This paper presents a phase-resolved optical coherence tomography (OCT) system that uses the polarization quadrature encoding method in a two-channel Mach-Zehnder interferometer. OCT is a powerful optical signal acquisition method that can capture depth-resolved micrometer-resolution images. In our method, a complex signal is optically generated, and its real and imaginary components are encoded in the orthogonal polarization states of one sample beam; absolute phase information can then be acquired instantaneously. Neither phase modulation nor numerical Fourier or Hilbert transformation to extract phase information is required, thereby decreasing data acquisition rates and processing time. We conducted signal post-processing to select data from the instabilities of reference scanning delay lines; the measured phase sensitivity was as low as 0.23 - , and the corresponding path-difierence resolution was 265pm. A localized surface proflle measurement of a chromium- coated layer deposited on a commercial resolution target surface was conducted. The results conflrmed that successful images can be obtained even with very small optical path difierences using the proposed method.

Charge transport in polycrystalline and single crystal synthetic diamond using ion beam induced charge imaging

Ion beam induced charge (IBIC) imaging has been used to investigate charge transport in high quality polycrystalline CVD diamond and single crystal synthetic diamond. In polycrystalline material the intra-crystallite charge transport within single crystals was studied. Micrometer resolution images of charge signal amplitude in different regions of a diamond crystallite are obtained as a function of the electric field strength. The charge signal amplitude, which is directly related to charge carrier drift length, is correlated to the distribution of radiative centres in the diamond identified using cathodoluminescence imaging. By comparison, IBIC imaging of single crystal synthetic diamond was also carried out. The sample, which contained deliberately introduced nitrogen lines, showed electron and hole CCE values close to 100%, in the low nitrogen regions. By comparison with CL and UV luminescence images, direct evidence is observed for the role of nitrogen and dislocations as deep trapping centres and their reduction of both electron and hole lifetimes.