High-resolution 3D Reconstruction Research Articles

Detailed Mapping of Hydrothermal Vent Fauna: A 3D Reconstruction Approach Based on Video Imagery

Active hydrothermal vent fields are complex, small-scale habitats hosting endemic fauna that changes at scales of centimeters, influenced by topographical variables. In previous studies, it has been shown that the distance to hydrothermal fluids is also a major structuring factor. Imagery analysis based on two dimensional photo stitching revealed insights to the vent field zonation around fluid exits and a basic knowledge of faunal assemblages within hydrothermal vent fields. However, complex three dimensional surfaces could not be adequately replicated in those studies, and the assemblage structure, as well as their relation to abiotic terrain variables, is often only descriptive. In this study we use ROV video imagery of a hydrothermal vent field on the southeastern Indian Ridge in the Indian Ocean. Structure from Motion photogrammetry was applied to build a high resolution 3D reconstruction model of one side of a newly discovered active hydrothermal chimney complex, allowing for the quantification of abundances. Likewise, the reconstruction was used to infer terrain variables at a scale important for megabenthic specimens, which were related to the abundances of the faunal assemblages. Based on the terrain variables, applied random forest model predicted the faunal assemblage distribution with an accuracy of 84.97 %. The most important structuring variables were the distances to diffuse- and black fluid exits, as well as the height of the chimney complex. This novel approach enabled us to classify quantified abundances of megabenthic taxa to distinct faunal assemblages and relate terrain variables to their distribution. The successful prediction of faunal assemblage occurrences further supports the importance of abiotic terrain variables as key structuring factors in hydrothermal systems and offers the possibility to detect suitable areas for Marine Protected areas on larger spatial scales. This technique works for any kind of video imagery, regardless of its initial purpose and can be implemented in marine monitoring and management.

First 3D imaging characterization of Pele\u2019s hair from Kilauea volcano (Hawaii)

In this work the morphologic features of Pele’s hair formed during three different eruptions of Kilauea volcano have been investigated: fountaining from Kilauea Iki’s 1959 Episode 1, weak explosive activity from Halemaumau lava lake and littoral explosions at Waikupanaha (2009). Morphological studies were performed by optical, stereo- and scanning electron microscopy. For the first time 3D image analysis was carried out by synchrotron radiation X-ray computed microtomography, which allowed a high-resolution 3D reconstruction of the internal structure of each Pele’s hair, highlighting several differences in terms of number density, elongation and shape of the vesicles between the samples from the three eruptions. We identified three main parameters determining these differences: initial size of the magma droplet, ejection velocity and magma viscosity. Pele’s hair erupted during the Kilauea Iki’s fountaining shows the highest thickness and the least elongated shape of the vesicles, though it is related to fast ejection of a low viscosity magma. We therefore suggest that the size of magma droplets is the main parameter influencing the morphology and inner textures of the Pele’s hair. The comparison with Pele’s hair of similar eruptions elsewhere demonstrates that there is no univocal correspondence between eruptive style and Pele’s hair texture.

Enhancing the X-ray contrast of polymeric biochromatography particles for three-dimensional imaging

A major limitation of the three-dimensional imaging of polymeric biochromatography particle packings using X-ray computed tomography is that the particles have a low density and a high porosity, making them almost undistinguishable from the surrounding liquid phase. Additionally, the employed media are typically composed of materials with low atomic numbers, which exhibit low X-ray absorption. We report an improvement of packed column reconstruction using micro X-ray computed tomography. A simple, inexpensive, and fast method to increase the contrast factor of highly porous polymer-based chromatographic particles was developed by applying a modified pore-blocking method. This approach relies on the selective filling of the porous chromatographic particles with a hydrophilic phase while a hydrophobic phase occupies the void spaces between the particles. The hydrophilic phase contains a dissolved X-ray absorbing radiocontrast agent. No chemical modifications of the chromatographic beads or columns were necessary. The developed method can be applied in-situ in a previously packed column and can be used for media with different organic backbones. We show the applicability of this method by carrying out the first 3D-reconstruction of packed micro columns with an inner diameter of 760 μm. The micro column contained agarose- and methacrylate-based particles commonly used in preparative biochromatography with mean diameters of 40 and 65 μm, respectively. Based on the obtained high-resolution 3D-reconstructions, we exemplarily computed packing properties such as global extraparticle porosity and radial porosity profiles, and visualized the presence of void spaces using 3D image analysis.

Aerial path planning for urban scene reconstruction

Small unmanned aerial vehicles (UAVs) are ideal capturing devices for high-resolution urban 3D reconstructions using multi-view stereo. Nevertheless, practical considerations such as safety usually mean that access to the scan target is often only available for a short amount of time, especially in urban environments. It therefore becomes crucial to perform both view and path planning to minimize flight time while ensuring complete and accurate reconstructions. In this work, we address the challenge of automatic view and path planning for UAV-based aerial imaging with the goal of urban reconstruction from multi-view stereo. To this end, we develop a novel continuous optimization approach using heuristics for multi-view stereo reconstruction quality and apply it to the problem of path planning. Even for large scan areas, our method generates paths in only a few minutes, and is therefore ideally suited for deployment in the field. To evaluate our method, we introduce and describe a detailed benchmark dataset for UAV path planning in urban environments which can also be used to evaluate future research efforts on this topic. Using this dataset and both synthetic and real data, we demonstrate survey-grade urban reconstructions with ground resolutions of 1 cm or better on large areas (30 000 m 2 ).

High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique we developed, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope set-up utilising an improved light-sheet generator allows us to visualise long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applying image fusion from orthogonal directions. This methodological integration of novel chemical, optical, and computational techniques allows a major advance in the analysis of global neural circuit organisation.

Outcrop-scale tomography: Insights into the 3D structure of multiphase boudins

We analyze the 3D geomorphology of a boudinaged amphibolite layer encased in marble using meter-scale tomography by serial sectioning, high resolution imaging and 3D reconstruction of a 2 m³ block from a quarry in the high-grade core of the Naxos core complex. Local uncertainties in the model are resolved by dissolution of marble in selected slabs, yielding a large, highly accurate 3D model.Results show five generations of brittle extension of the amphibolite in ductile marble, although examination of the boudinaged layer on the outside of the block only suggested one generation. Most prominent are the rotated domino boudins, where part of the dilatancy between domino boudins can be shown to be due to later reactivation. Shear fractures between domino boudins have relatively large throw gradients, indicating ductile rupture. Layer-parallel stretch as measured by reconstruction of domino boudins decreases with amphibolite layer thickness although the layer has undergone the same bulk stretch everywhere. This suggests that with decreasing layer thickness deformation becomes delocalized, offering tantalizing glimpses of rheology under metamorphic conditions.

Volta phase plate data collection facilitates image processing and cryo-EM structure determination.

A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.

GENFIRE: A generalized Fourier iterative reconstruction algorithm for high-resolution 3D imaging

Tomography has made a radical impact on diverse fields ranging from the study of 3D atomic arrangements in matter to the study of human health in medicine. Despite its very diverse applications, the core of tomography remains the same, that is, a mathematical method must be implemented to reconstruct the 3D structure of an object from a number of 2D projections. Here, we present the mathematical implementation of a tomographic algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE), for high-resolution 3D reconstruction from a limited number of 2D projections. GENFIRE first assembles a 3D Fourier grid with oversampling and then iterates between real and reciprocal space to search for a global solution that is concurrently consistent with the measured data and general physical constraints. The algorithm requires minimal human intervention and also incorporates angular refinement to reduce the tilt angle error. We demonstrate that GENFIRE can produce superior results relative to several other popular tomographic reconstruction techniques through numerical simulations and by experimentally reconstructing the 3D structure of a porous material and a frozen-hydrated marine cyanobacterium. Equipped with a graphical user interface, GENFIRE is freely available from our website and is expected to find broad applications across different disciplines.

JAK/STAT signaling is necessary for cell monosis prior to epithelial cell apoptotic extrusion

Epithelial cell extrusion is crucial for proper development and tissue homeostasis. High-resolution 3D reconstruction and 4D imaging, combined with genetic analyis, have allowed us to reveal the highly-sterotyped morphogenetic events controlled by JAK/STAT signaling in a developmentally-programmed case of epithelial cell extrusion. Specialized somatic cells, Polar Cells (PCs), are produced in excess and then undergo apoptotic elimination from the follicular epithelium in the Drosophila ovary. We show that supernumerary PCs are first systematically enveloped by PC neighbors on all sides, first laterally, then apically in conjunction with highly-reinforced adherens junctions, and finally basally. The PC to be removed thus loses all contact with follicle cells, germline cells and the basement membrane in a process we have called cell ‘monosis’, for ‘isolation’ in Greek. PC monosis takes several hours, and always precedes, and is independent of, activation of apoptosis. JAK/STAT signaling is necessary within the surrounding follicular epithelium for PC monosis. Minutes after monosis is complete, PC apoptotic corpses are formed and extruded laterally within the epithelium, in contrast to the apical and basal extrusions described to date. These apoptotic corpses are engulfed and eliminated by surrounding follicle cells, which are thus acting as non-professional phagocytes. This study therefore shows the non cell-autonomous impact of an epithelium, via JAK/STAT signaling activation, on cell morphogenesis events leading to apoptotic extrusion. It is likely that the use of high-resolution 3D and 4D imaging, which allows for better spatio-temporal understanding of morphogenetic events, will reveal that cell monosis and lateral extrusion within an epithelium are pertinent for other cases of epithelial cell extrusion as well.

A simple, fast, and repeatable survey method for underwater visual 3D benthic mapping and monitoring

Visual 3D reconstruction techniques provide rich ecological and habitat structural information from underwater imagery. However, an unaided swimmer or diver struggles to navigate precisely over larger extents with consistent image overlap needed for visual reconstruction. While underwater robots have demonstrated systematic coverage of areas much larger than the footprint of a single image, access to suitable robotic systems is limited and requires specialized operators. Furthermore, robots are poor at navigating hydrodynamic habitats such as shallow coral reefs. We present a simple approach that constrains the motion of a swimmer using a line unwinding from a fixed central drum. The resulting motion is the involute of a circle, a spiral‐like path with constant spacing between revolutions. We test this survey method at a broad range of habitats and hydrodynamic conditions encircling Lizard Island in the Great Barrier Reef, Australia. The approach generates fast, structured, repeatable, and large‐extent surveys (~110 m2 in 15 min) that can be performed with two people and are superior to the commonly used “mow the lawn” method. The amount of image overlap is a design parameter, allowing for surveys that can then be reliably used in an automated processing pipeline to generate 3D reconstructions, orthographically projected mosaics, and structural complexity indices. The individual images or full mosaics can also be labeled for benthic diversity and cover estimates. The survey method we present can serve as a standard approach to repeatedly collecting underwater imagery for high‐resolution 2D mosaics and 3D reconstructions covering spatial extents much larger than a single image footprint without requiring sophisticated robotic systems or lengthy deployment of visual guides. As such, it opens up cost‐effective novel observations to inform studies relating habitat structure to ecological processes and biodiversity at scales and spatial resolutions not readily available previously.

Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular Cell Network.

Secondary lymphoid organs have developed segregated niches that are able to initiate and maintain effective immune responses. Such global organization requires tight control of diverse cellular components, specifically those that regulate lymphocyte trafficking. Fibroblastic reticular cells (FRCs) form a densely interconnected network in lymph nodes and provide key factors necessary for T cell migration and retention, and foster subsequent interactions between T cells and dendritic cells. Development of integrative systems biology approaches has made it possible to elucidate this multilevel complexity of the immune system. Here, we present a graph theory-based analysis of the FRC network in murine lymph nodes, where generation of the network topology is performed using high-resolution confocal microscopy and 3D reconstruction. This approach facilitates the analysis of physical cell-to-cell connectivity, and estimation of topological robustness and global behavior of the network when it is subjected to perturbation in silico.

The 4-Dimensional Plant: Effects of Wind-Induced Canopy Movement on Light Fluctuations and Photosynthesis.

Physical perturbation of a plant canopy brought about by wind is a ubiquitous phenomenon and yet its biological importance has often been overlooked. This is partly due to the complexity of the issue at hand: wind-induced movement (or mechanical excitation) is a stochastic process which is difficult to measure and quantify; plant motion is dependent upon canopy architectural features which, until recently, were difficult to accurately represent and model in 3-dimensions; light patterning throughout a canopy is difficult to compute at high-resolutions, especially when confounded by other environmental variables. Recent studies have reinforced the expectation that canopy architecture is a strong determinant of productivity and yield; however, links between the architectural properties of the plant and its mechanical properties, particularly its response to wind, are relatively unknown. As a result, biologically relevant data relating canopy architecture, light- dynamics, and short-scale photosynthetic responses in the canopy setting are scarce. Here, we hypothesize that wind-induced movement will have large consequences for the photosynthetic productivity of our crops due to its influence on light patterning. To address this issue, in this study we combined high resolution 3D reconstructions of a plant canopy with a simple representation of canopy perturbation as a result of wind using solid body rotation in order to explore the potential effects on light patterning, interception, and photosynthetic productivity. We looked at two different scenarios: firstly a constant distortion where a rice canopy was subject to a permanent distortion throughout the whole day; and secondly, a dynamic distortion, where the canopy was distorted in incremental steps between two extremes at set time points in the day. We find that mechanical canopy excitation substantially alters light dynamics; light distribution and modeled canopy carbon gain. We then discuss methods required for accurate modeling of mechanical canopy excitation (here coined the 4-dimensional plant) and some associated biological and applied implications of such techniques. We hypothesize that biomechanical plant properties are a specific adaptation to achieve wind-induced photosynthetic enhancement and we outline how traits facilitating canopy excitation could be used as a route for improving crop yield.

High-resolution 3D reconstruction of microtubule structures by quantitative multi-angle total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve 40nm axial resolution in experimental conditions where SNR is as low as 2, with ~35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.

Endoscopic-CT: Learning-Based Photometric Reconstruction for Endoscopic Sinus Surgery.

In this work we present a method for dense reconstruction of anatomical structures using white light endoscopic imagery based on a learning process that estimates a mapping between light reflectance and surface geometry. Our method is unique in that few unrealistic assumptions are considered (i.e., we do not assume a Lambertian reflectance model nor do we assume a point light source) and we learn a model on a per-patient basis, thus increasing the accuracy and extensibility to different endoscopic sequences. The proposed method assumes accurate video-CT registration through a combination of Structure-from-Motion (SfM) and Trimmed-ICP, and then uses the registered 3D structure and motion to generate training data with which to learn a multivariate regression of observed pixel values to known 3D surface geometry. We demonstrate with a non-linear regression technique using a neural network towards estimating depth images and surface normal maps, resulting in high-resolution spatial 3D reconstructions to an average error of 0.53mm (on the low side, when anatomy matches the CT precisely) to 1.12mm (on the high side, when the presence of liquids causes scene geometry that is not present in the CT for evaluation). Our results are exhibited on patient data and validated with associated CT scans. In total, we processed 206 total endoscopic images from patient data, where each image yields approximately 1 million reconstructed 3D points per image.

High-Resolution Cryo-EM Studies on the Yeast Mitotic Kinesin-5

The kinesin-5 family is required for the assembly and maintenance of the bipolar organization of microtubules in the mitotic spindle. Recently, the yeast kinesin-5 (Cin8) was shown to exhibit unusual directional properties. Previously, all kinesins were thought to act exclusively as plus-end or minus-end directed, depending on the location of the motor domain. Despite possessing an N-terminal motor like conventional kinesin-1, the yeast kinesin-5 Cin8 acts as a minus-end directed motor when working individually on single microtubules. Furthermore, Cin8 is the only known kinesin with the ability to switch directions when working in a team crosslinking two anti-parallel microtubules. Thus, the accumulation of Cin8 at the spindle poles located at the minus-end is thought to trigger a directional switch to plus-end directed motility, which is required for the bidirectional sliding of anti-parallel microtubules. Deletion of a unique insert in loop 8 (L8) found only in Cin8 induces a strong bias towards minus-end directed motility, suggesting an important role in enforcing the switch to plus-end directed. To investigate the mechanistic basis for the unusual properties of Cin8, we are using cryo-EM, which uniquely allows for high-resolution 3D reconstructions of Cin8 when bound to microtubules. Initial efforts have yielded a ∼7A reconstruction in the ADP-AlFx state, which revealed Cin8-specific differences in the domain structure, such as the extended L8. We are pursuing higher resolution structures of this and other nucleotide states in order to reveal how Cin8 is able to achieve minus-end directed motility despite being an N-terminal kinesin.

Whole Transcriptome Screening Reveals Myelination Deficits in Dysplastic Human Temporal Neocortex.

Focal cortical dysplasias (FCDs) are local malformations of the human neocortex with strong epileptogenic potential. To investigate the underlying pathomechanisms, we performed a whole human transcriptome screening to compare the gene expression pattern of dysplastic versus nondysplastic temporal neocortex. Tissue obtained from FCD IIIa cases (mean age 20.5 years) who had undergone surgical treatment, due to intractable epilepsy, was compared with nondysplastic specimens (mean age 19.9 years) by means of Affymetrix arrays covering 28 869 genes. We found 211 differentially expressed genes (DEX) among which mainly genes important for oligodendrocyte differentiation and myelination were downregulated in FCD IIIa. These findings were confirmed as functionally important by Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis. The reduced expression of myelin-associated transcripts was confirmed for FCD Ia, IIa, and IIIa by real-time RT-qPCR. In addition, we found that the density of myelin basic protein mRNA-expressing oligodendrocytes and of 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive myelin fibers was significantly reduced in dysplastic cortex. Moreover, high-resolution confocal imaging and 3D reconstruction revealed that the myelin fiber network was severely disorganized in dysplastic neocortex, indicating a disturbance of myelin sheath formation and maintenance in FCD.

2.9 Å Resolution Cryo-EM 3D Reconstruction of Close-Packed Virus Particles

Single-particle cryoelectron microscopy typically discards close-packed particle images as unusable data. Here, we report an image processing strategy and case study of obtaining near-atomic resolution 3D reconstructions from close-packed particles. Multiple independent de novo initial models were constructed to determine and cross-validate the particle parameters. The particles with consistent views were further refined including not only Euler angles and center positions but also defocus, astigmatism, beam tilt, and overall and anisotropic magnification. We demonstrated this strategy with a 2.9Å resolution reconstruction of a 1.67 MDa virus-like particle of a circovirus, PCV2, recorded on 86 photographic films. The map resolution was further validated with a phase-randomization test and local resolution assessment, and the atomic model was validated with MolProbity and EMRinger. Close-packed virus particles were thus shown not only to be useful for high-resolution 3D reconstructions but also to allow data collection at significantly improved throughput for near-atomic resolution reconstructions.

Glycinergic Neurotransmission: A Potent Regulator of Embryonic Motor Neuron Dendritic Morphology and Synaptic Plasticity.

MNs within the brainstem and spinal cord are responsible for integrating a diverse array of synaptic inputs into discrete contractions of skeletal muscle to achieve coordinated behaviors, such as breathing, vocalization, and locomotion. The last trimester in utero is critical in neuromotor development, as this is when central and peripheral synaptic connections are made onto and from MNs. At this time-point, using transgenic mice with negligible glycinergic postsynaptic responses, we show that this deficiency leads to abnormally high excitatory neurotransmission and alters the dendritic architecture responsible for coherently integrating these inputs. This study compliments the emerging concept that neurodevelopmental disorders (including autism, epilepsy, and amyotrophic lateral sclerosis) are underpinned by synaptic dysfunction and therefore will be useful to neuroscientists and neurologists alike.

4D Tumorigenesis Model for Quantitating Coalescence, Directed Cell Motility and Chemotaxis, Identifying Unique Cell Behaviors, and Testing Anticancer Drugs.

A 4D high-resolution computer-assisted reconstruction and motion analysis system has been developed and applied to the long-term (14-30 days) analysis of cancer cells migrating and aggregating within a 3D matrix. 4D tumorigenesis models more closely approximate the tumor microenvironment than 2D substrates and, therefore, are improved tools for elucidating the interactions within the tumor microenvironment that promote growth and metastasis. The model we describe here can be used to analyze the growth of tumor cells, aggregate coalescence, directed cell motility and chemotaxis, matrix degradation, the effects of anticancer drugs, and the behavior of immune and endothelial cells mixed with cancer cells. The information given in this chapter is also intended to acquaint the reader with computer-assisted methods and algorithms that can be used for high-resolution 3D reconstruction and quantitative motion analysis.

A fast cross-validation method for alignment of electron tomography images based on Beer-Lambert law.

In electron tomography, accurate alignment of tilt series is an essential step in attaining high-resolution 3D reconstructions. Nevertheless, quantitative assessment of alignment quality has remained a challenging issue, even though many alignment methods have been reported. Here, we report a fast and accurate method, tomoAlignEval, based on the Beer-Lambert law, for the evaluation of alignment quality. Our method is able to globally estimate the alignment accuracy by measuring the goodness of log-linear relationship of the beam intensity attenuations at different tilt angles. Extensive tests with experimental data demonstrated its robust performance with stained and cryo samples. Our method is not only significantly faster but also more sensitive than measurements of tomogram resolution using Fourier shell correlation method (FSCe/o). From these tests, we also conclude that while current alignment methods are sufficiently accurate for stained samples, inaccurate alignments remain a major limitation for high resolution cryo-electron tomography.