Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs^1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.     

A Reference-Free Method for Brightness Compensation and Contrast Enhancement of Micrographs of Serial Sections

Three-dimensional (3D) reconstruction of an organ or tissue from a stack of histologic serial sections provides valuable morphological information. The procedure includes section preparation of the organ or tissue, micrographs acquisition, image registration, 3D reconstruction, and visualization. However, the brightness and contrast through the image stack may not be consistent due to imperfections in the staining procedure, which may cause difficulties in micro-structure identification using virtual sections, region segmentation, automatic target tracing, etc. In the present study, a reference-free method, Sequential Histogram Fitting Algorithm (SHFA), is therefore developed for adjusting the severe and irregular variance of brightness and contrast within the image stack. To apply the SHFA, the gray value histograms of individual images are first calculated over the entire image stack and a set of landmark gray values are chosen. Then the histograms are transformed so that there are no abrupt changes in progressing through the stack. Finally, the pixel gray values of the original images are transformed into the desired ones based on the relationship between the original and the transformed histograms. The SHFA is tested on an image stacks from mouse kidney sections stained with toluidine blue, and captured by a slide scanner. As results, the images through the entire stack reveal homogenous brightness and consistent contrast. In addition, subtle color differences in the tissue are well preserved so that the morphological details can be recognized, even in virtual sections. In conclusion, compared with the existing histogram-based methods, the present study provides a practical method suitable for compensating brightness, and improving contrast of images derived from a large number of serial sections of biological organ.     

Facilitating tumor functional assessment by spatially relating 3D tumor histology and in vivo MRI: image registration approach

Background

Magnetic resonance imaging (MRI), together with histology, is widely used to diagnose and to monitor treatment in oncology. Spatial correspondence between these modalities provides information about the ability of MRI to characterize cancerous tissue. However, registration is complicated by deformations during pathological processing, and differences in scale and information content.

Methodology/Principal Findings

This study proposes a methodology for establishing an accurate 3D relation between histological sections and high resolution in vivo MRI tumor data. The key features of the methodology are: 1) standardized acquisition and processing, 2) use of an intermediate ex vivo MRI, 3) use of a reference cutting plane, 4) dense histological sampling, 5) elastic registration, and 6) use of complete 3D data sets. Five rat pancreatic tumors imaged by T2*-w MRI were used to evaluate the proposed methodology. The registration accuracy was assessed by root mean squared (RMS) distances between manually annotated landmark points in both modalities. After elastic registration the average RMS distance decreased from 1.4 to 0.7 mm. The intermediate ex vivo MRI and the reference cutting plane shared by all three 3D images (in vivo MRI, ex vivo MRI, and 3D histology data) were found to be crucial for the accurate co-registration between the 3D histological data set and in vivo MRI. The MR intensity in necrotic regions, as manually annotated in 3D histology, was significantly different from other histologically confirmed regions (i.e., viable and hemorrhagic). However, the viable and the hemorrhagic regions showed a large overlap in T2^*-w MRI signal intensity.

Conclusions

The established 3D correspondence between tumor histology and in vivo MRI enables extraction of MRI characteristics for histologically confirmed regions. The proposed methodology allows the creation of a tumor database of spatially registered multi-spectral MR images and multi-stained 3D histology.     

Prolonged in vivo functional assessment of the mouse oviduct using optical coherence tomography through a dorsal imaging window

The oviduct (or fallopian tube) serves as an environment for gamete transport, fertilization and preimplantation embryo development in mammals. Although there has been increasing evidence linking infertility with disrupted oviduct function, the specific roles that the oviduct plays in both normal and impaired reproductive processes remain unclear. The mouse is an important mammalian model to study human reproduction. However, most of the current analyses of the mouse oviduct rely on static histology or 2D visualization, and are unable to provide dynamic and volumetric characterization of this organ. The lack of imaging access prevents longitudinal live analysis of the oviduct and its associated reproductive events, limiting the understanding of mechanistic aspects of fertilization and preimplantation pregnancy. To address this limitation, we report a 3D imaging approach that enables prolonged functional assessment of the mouse oviduct in vivo. By combining optical coherence tomography with a dorsal imaging window, this method allows for extended volumetric visualization of the oviduct dynamics, which was previously not achievable. The approach is used for quantitative analysis of oviduct contraction, spatiotemporal characterization of cilia beat frequency and longitudinal imaging. This new approach is a useful in vivo imaging platform for a variety of live studies in mammalian reproduction.      

Focus gradient correction applied to tilt series image data used in electron tomography

The resolution in 3D reconstructions from tilt series is limited to the information below the first zero of the contrast transfer function unless the signal is corrected computationally. The restoration is usually based on the assumption of a linear space-invariant system and a linear relationship between object mass density and observed image contrast. The space-invariant model is no longer valid when applied to tilted micrographs because the defocus varies in a direction perpendicular to the tilt axis and with it the shape of the associated point spread function. In this paper, a method is presented for determining the defocus gradient in thin specimens such as sections and 2D crystals, and for restoration of the images subsequently used for 3D reconstruction. The alignment procedure for 3D reconstruction includes area matching and tilt geometry refinement. A map with limited resolution computed from uncorrected micrographs is compared to a volume computed from corrected micrographs with extended resolution.     

The restoration of electron micrographs blurred by drift and rotation

We have investigated the restoration of electron micrographs exhibiting blurring due to drift and rotation. Blurring due to drift arises in micrographs taken of a specimen which is moving relative to the image plane. A related problem is that of rotational blurring which arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis. Restoration algorithms were evaluated by applying them to the restoration of blurred model images degraded by additive Gaussian noise. Model images were also used to investigate how an incorrect estimate of the point spread function describing the blur would effect the restoration. Images were, if necessary, geometrically transformed to a space in which the point spread function of the blur can be considered as linear and space invariant as, under these conditions, the restoration algorithms are greatly simplified. In the case of the rotationally blurred images this procedure was accomplished by transforming the image to polar coordinates. The restoration techniques were successfully applied to blurred micrographs of bacteriophage T4 and crystals of catalase. The quality of the restoration was judged by comparisons of the restored images to undegraded images. Application to micrographs of rotationally blurred cross sections of helical macrofibers of sickle hemoglobin resulted in a reduction in the amount of rotational blurring.     

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

Background

Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.

Methods

FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.

Results

The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.

Conclusion

We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.     

Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy

Three-dimensional electron microscopy(3 D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3 D reconstruction of biological samples remains challenging. Herein, we present a 3 D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and38.57 nm lateral resolution using AutoCUTS-SEM(automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P.wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.     

TMaCS: A hybrid template matching and classification system for partially-automated particle selection

Selection of particle images from electron micrographs presents a bottleneck in determining the structures of macromolecular assemblies by single particle electron cryomicroscopy (cryo-EM). The problem is particularly important when an experimentalist wants to improve the resolution of a 3D map by increasing by tens or hundreds of thousands of images the size of the dataset used for calculating the map. Although several existing methods for automatic particle image selection work well for large protein complexes that produce high-contrast images, it is well known in the cryo-EM community that small complexes that give low-contrast images are often refractory to existing automated particle image selection schemes. Here we develop a method for partially-automated particle image selection when an initial 3D map of the protein under investigation is already available. Candidate particle images are selected from micrographs by template matching with template images derived from projections of the existing 3D map. The candidate particle images are then used to train a support vector machine, which classifies the candidates as particle images or non-particle images. In a final step in the analysis, the selected particle images are subjected to projection matching against the initial 3D map, with the correlation coefficient between the particle image and the best matching map projection used to assess the reliability of the particle image. We show that this approach is able to rapidly select particle images from micrographs of a rotary ATPase, a type of membrane protein complex involved in many aspects of biology.     

Banking of fresh-frozen prostate tissue using the alternate mirror image protocol: methods, validation, and impact on the pathological prognostic parameters in radical prostatectomy

We evaluated the value of the ‘alternative slices mirror image method’ used in prostate tissue banking in terms of predicting the sampling of cancerous tissue while preserving the pathological prognostic information. The concordance of diagnosis between banked sections and their mirror image paraffin- sections was studied using 50 cases corresponding to 400 H&E sections taken from 400 banked frozen blocks (two presumed benign and two presumed cancer for each case). The mean number of paraffin blocks in each case was 21. On average 29% of the prostate gland was banked and banked tissue contained cancer in 47 cases (94%). There was no difference between the concordant and discordant groups in terms of the final Gleason score, pathological stage, prostate size, number of banked blocks and the percentage of the prostate submitted for banking. However, concordant cases had larger foci of cancer in the mirror image paraffin block (P?=?0.0088). In addition, the surgical margins sections which are not banked using this method provided important information about the pathological stage, surgical margins status and the final Gleason score in 2.6, 2.6, and 1.3% of cases, respectively. The ‘alternative slices mirror image method’ is a straightforward method that is highly efficient in banking prostatic cancerous tissue. Overall, tumor volume and especially size of tumor foci in the image paraffin block are the most important factors in dictating the success rate of banking frozen cancerous tissue. Including ‘surgical margins’ sections for histology provides additional important prognostic information in a minority of cases.     

Film tomography as a tool for three-dimensional image construction and gene expression studies

In order to observe three-dimensional (3D) expression patterns of genes in whole animals, whole organs, or whole tissues, in situ hybridization (ISH) of many sections must be carried out and then used to construct a 3D image. For this purpose, we have developed an automatic microtome to prepare tissue sections with an adhesive film. We used commercially available film suitable for sectioning and ISH. We constructed a microtome and, after adherence of the film to a paraffin-embedded tissue block, cut the block with a blade to prepare sections on film. Then, the sections-on-film were automatically set in a plastic frame that was the same size as a conventional glass slide. With this automatic microtome, tissue sections can be made for ISH or immunohistochemistry in addition to conventional hematoxylin and eosin staining without specific training. We demonstrate that we can construct 3D images of gene expression patterns obtained by ISH on sections prepared with this automatic microtome. We have designated this method as 'Film Tomography (FITO)'.     

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead’s position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 μm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.     

A Method for 3D Histopathology Reconstruction Supporting Mouse Microvasculature Analysis

Structural abnormalities of the microvasculature can impair perfusion and function. Conventional histology provides good spatial resolution with which to evaluate the microvascular structure but affords no 3-dimensional information; this limitation could lead to misinterpretations of the complex microvessel network in health and disease. The objective of this study was to develop and evaluate an accurate, fully automated 3D histology reconstruction method to visualize the arterioles and venules within the mouse hind-limb. Sections of the tibialis anterior muscle from C57BL/J6 mice (both normal and subjected to femoral artery excision) were reconstructed using pairwise rigid and affine registrations of 5 µm-thick, paraffin-embedded serial sections digitized at 0.25 µm/pixel. Low-resolution intensity-based rigid registration was used to initialize the nucleus landmark-based registration, and conventional high-resolution intensity-based registration method. The affine nucleus landmark-based registration was developed in this work and was compared to the conventional affine high-resolution intensity-based registration method. Target registration errors were measured between adjacent tissue sections (pairwise error), as well as with respect to a 3D reference reconstruction (accumulated error, to capture propagation of error through the stack of sections). Accumulated error measures were lower (p<0.01) for the nucleus landmark technique and superior vasculature continuity was observed. These findings indicate that registration based on automatic extraction and correspondence of small, homologous landmarks may support accurate 3D histology reconstruction. This technique avoids the otherwise problematic “banana-into-cylinder” effect observed using conventional methods that optimize the pairwise alignment of salient structures, forcing them to be section-orthogonal. This approach will provide a valuable tool for high-accuracy 3D histology tissue reconstructions for analysis of diseased microvasculature.     

Rapid Particle Size Measurement Using 3D Surface Imaging

The present study introduces a new three-dimensional (3D) surface image analysis technique in which white light illumination from different incident angles is used to create 3D surfaces with a photometric approach. The three-dimensional features of the surface images created are then used in the characterization of particle size distributions of granules. This surface image analysis method is compared to sieve analysis and a particle sizing method based on spatial filtering technique with nearly 30 granule batches. The aim is also to evaluate the technique in flowability screening of granular materials. Overall, the new 3D imaging approach allows a rapid analysis of large amounts of sample and gives valuable visual information on the granule surfaces in terms of surface roughness and particle shape.     

A methodology for registration of a histological slide and in vivo MRI volume based on optimizing mutual information

We present a method for registering histology and in vivo imaging that requires minimal microtoming and is automatic following the user's initialization. In this demonstration, we register a single hematoxylin-and-eosin-stained histological slide of a coronal section of a rat brain harboring a 9L gliosarcoma with an in vivo 7T MR image volume of the same brain. Because the spatial resolution of the in vivo MRI is limited, we add the step of obtaining a high spatial resolution, ex vivo MRI in situ for intermediate registration. The approach taken was to maximize mutual information in order to optimize the registration between all pairings of image data whether the sources are MRI, tissue block photograph, or stained sample photograph. The warping interpolant used was thin plate splines with the appropriate basis function for either 2-D or 3-D applications. All registrations were implemented by user initialization of the approximate pose between the two data sets, followed by automatic optimization based on maximizing mutual information. Only the higher quality anatomical images were used in the registration process; however, the spatial transformation was directly applied to a quantitative diffusion image. Quantitative diffusion maps from the registered location appeared highly correlated with the H&E slide. Overall, this approach provides a robust method for coregistration of in vivo images with histological sections and will have broad applications in the field of functional and molecular imaging.     

Three-dimensional registration of histology of human atherosclerotic carotid plaques to in-vivo imaging

An accurate spatial relationship between 3D in-vivo carotid plaque and lumen imaging and histological cross sections is required to study the relationship between biomechanical parameters and atherosclerotic plaque components. We present and evaluate a fully three-dimensional approach for this registration problem, which accounts for deformations that occur during the processing of the specimens. By using additional imaging steps during tissue processing and semi-automated non-linear registration techniques, a 3D-reconstruction of the histology is obtained.The methodology was evaluated on five specimens obtained from patients, operated for severe atherosclerosis in the carotid bifurcation. In more than 80% of the histology slices, the quality of the semi-automated registration with computed tomography angiography (CTA) was equal to or better than the manual registration. The inter-observer variability was between one and two in-vivo CT voxels and was equal to the manual inter-observer variability. Our technique showed that the angles between the normals of the registered histology slices and the in-vivo CTA scan direction ranged 6–56°, indicating that proper 3D-registration is crucial for establishing a correct spatial relation with in-vivo imaging modalities. This new 3D-reconstruction technique of atherosclerotic plaque tissue opens new avenues in the field of biomechanics as well as in the field of image processing, where it can be used for validation purposes of segmentation algorithms.     

Three-dimensional computer reconstructions from serial sections of cell nuclei

The analysis of ultrathin serial sections as 3-dimensional (3D) information requires interpretation and display of a large amount of data. We suggest a simple way to solve this problem; it permits presentation of a series of sections as a 3D color image of good quality. It involves a picture system with specialized hardware and software written for this purpose. 3D images of cellular organelles have been drawn either by manually defining the contour of the objects or by thresholding of the volumes in the structures. These 2 methods allow rapid drawing of the image on the screen. It is possible to determine the position, shape and size of 3D structures. This interactive system allows the user to choose between several options: colors, removal of parts of the object, and cutout.     

Automated correlation of single particle tilt pairs for Random Conical Tilt and Orthogonal Tilt Reconstructions

One of the major methodological challenges in single particle electron microscopy is obtaining initial reconstructions which represent the structural heterogeneity of the dataset. Random Conical Tilt and Orthogonal Tilt Reconstruction techniques in combination with 3D alignment and classification can be used to obtain initial low-resolution reconstructions which represent the full range of structural heterogeneity of the dataset. In order to achieve statistical significance, however, a large number of 3D reconstructions, and, in turn, a large number of tilted image pairs are required. The extraction of single particle tilted image pairs from micrographs can be tedious and time-consuming, as it requires intensive user input even for semi-automated approaches. To overcome the bottleneck of manual selection of a large number of tilt pairs, we developed an algorithm for the correlation of single particle images from tilted image pairs in a fully automated and user-independent manner. The algorithm reliably correlates correct pairs even from noisy micrographs. We further demonstrate the applicability of the algorithm by using it to obtain initial references both from negative stain and unstained cryo datasets.     

Two-photon confocal microscopy: a nondestructive method for studying wound healing

Two-photon confocal microscopy is a new technology useful in nondestructive analysis of tissue. The pattern generated from laser-excited autofluorescence and second harmonic signals can be analyzed to construct a three-dimensional, microanatomical, structural image. The healing of full-thickness guinea pig skin wounds was studied over a period of 28 days using two-photon confocal microscopy. Three-dimensional data were rendered from two-dimensional images and compared with conventional, en face, histologic sections. Two-photon confocal microscopy images show resolution of muscle, fascia fibers, collagen fibers, inflammatory cells, blood vessels, and hair. Although these images do not currently have the resolution of standard histology, the ability to noninvasively acquire three-dimensional images of skin promises to be an important tool in wound-healing studies.     

Finding paths in a labyrinth based on reaction-diffusion media.

During the past few decades, many proposals were made on how to take an effective solution for finding a path in a labyrinth, one of the most well known problems of high computational complexity inherent in information processing by biomolecular and biological entities. In particular, attempts were made to use a technique attractive enough for solving this problem based on wave processes in reaction-diffusion media. Trigger waves in reaction diffusion systems spread simultaneously through all paths of the labyrinth in a highly parallel mode. Regretfully, the velocity of these waves is extremely low which gave no way for the practical implementation of this technique until now. An effective 'hardware' system was designed which was capable of finding a path in a labyrinth using fast phase waves. Three principal points were assumed as a basis for this design, i.e. (1) hybrid architecture that combined an information processing reaction-diffusion medium which performs operations of high computational complexity with a digital computer carrying out supplementary image processing operations; (2) light-sensitive information processing media of Belousov-Zhabotinsky type that enables the simulation of the labyrinth and spreading wave evolution by their images stored in the medium and reduces the problem to the image processing operations; (3) fast light-induced phase wave processes that spreads through the labyrinth in several seconds instead of hours which is typical for trigger waves inherent in reaction-diffusion media. These fundamentals along with the additional procedure of testing for labyrinth fragment connectness provided us with the opportunity to solve labyrinth problems.