Three-dimensional microscopy data exploration by interactive volume visualization

This paper presents a new volume visualization approach for three-dimensional (3-D) interactive microscopy data exploration. Because of their unique image characteristics, 3-D microscopy data are often not able to be visualized effectively by conventional volume visualization techniques. In our approach, microscopy visualization is carried out in an interactive data exploration environment, based on a combination of interactive volume rendering techniques and image-based transfer function design methods. Interactive volume rendering is achieved by using two-dimensional (2-D) texture mapping in a Shear-Warp volume rendering algorithm. Image processing techniques are employed and integrated into the rendering pipeline for the definition and searching of appropriate transfer functions that best reflect the user's visualization intentions. These techniques have been implemented successfully in a prototype visualization system on low-end and middle-range SGI desktop workstations. Since only 2-D texture mapping is required, the system can also be easily ported to PC platforms.     

Three‐dimensional imaging of porous media using confocal laser scanning microscopy

In the last decade, imaging techniques capable of reconstructing three‐dimensional (3‐D) pore‐scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO₂ storage potential. CLSM has a unique capability of producing 3‐D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z‐dimension) that can be imaged in porous materials. In this study, we introduce a ‘grind and slice’ technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3‐D confocal volumetric data into pores and grains. Finally, we use the resulting 3‐D pore‐scale binarized confocal data obtained to quantitatively determine petrophysical pore‐scale properties such as total porosity, macro‐ and microporosity and single‐phase permeability using lattice Boltzmann (LB) simulations, validated by experiments.     

Three‐dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy

Three‐dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step‐by‐step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings.     

Three‐dimensional visualization of dermal skin structure using confocal laser scanning microscopy

The properties and performance of collagen‐based materials may be affected by the collagen fibre bundle pattern, orientation and weave. The aim of this study was to develop and apply methods to visualize the dermis using confocal laser scanning microscopy from thin tissue sections stained with haematoxylin and eosin. The data was processed to allow three‐dimensional (3‐D) visualization on a PC and using a 3‐D immersive technology system. The 3‐D visualization of the confocal microscope image stacks allowed the evaluation of the collagen macromolecular structure including the collagen fibre bundles. The methods developed provide a novel way of viewing complex organic structures with further potential applications in the medical field.     

Three-dimensional visualization methods for confocal microscopy

Three-dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three-dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi-transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.     

Three‐dimensional visualization of rat retina by X‐ray differential phase contrast tomographic microscopy

The retina is one of the most tiny and sophisticated tissues of the body. Three dimensional (3D) visualization of the whole retina is valuable both in clinical and research arenas. The tissue has been predominantly assessed by time‐consuming histopathology and optical coherence tomography (OCT) in research and clinical arenas. However, none of the two methods can provide 3D imaging of the retina. The purpose of this study is to give a volumetric visualization of rat retina at submicron resolution, using an emerging imaging technique‐phase‐contrast X‐ray CT. A Sprague‐Dawley (SD) rat eye specimen was scanned with X‐ray differential phase contrast tomographic microscopy (DPC‐microCT) equipped at the Swiss Light Source synchrotron. After scanning, the specimen was subjected to routine histology procedures and severed as a reference. The morphological characteristics and signal features of the retina in the DPC‐microCT images were evaluated. The total retina and its sublayers thicknesses were measured on the DPC‐microCT images and compared with those obtained from the histological sections. The retina structures revealed by DPC‐microCT were highly consistent with the histological section. In this study, we achieved nondestructive 3D visualization of SD rat retina. In addition to detailed anatomical structures, the objective parameters provided by DPC‐microCT make it a useful tool for retinal research and disease diagnosis in the early stage.     

Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best‐known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second‐harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second‐harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second‐harmonic generation, as a second‐order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser‐scanning microscope. In this work, we combine the axial resolving power of second‐harmonic generation and chiral sensitivity of second‐harmonic generation circular dichroism to realize three‐dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second‐harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second‐harmonic generation circular dichroism response in complicated three‐dimensional biological systems. The sample we use is starch granules whose second‐harmonic generation‐active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second‐harmonic generation for right‐handed circularly polarized excitation is significantly different from second‐harmonic generation for left‐handed one, offering excellent second‐harmonic generation circular dichroism contrast that approaches 100%. In addition, three‐dimensional visualization of second‐harmonic generation circular dichroism distribution with sub‐micrometer spatial resolution is realized. We observed second‐harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second‐harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three‐dimensional structures of starch granules. The second‐harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.     

Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

Visualization of volume data in confocal microscopy: comparison and improvements of volume rendering methods

Confocal microscopes routinely produce three-dimensional data sets. The visualization of these digital volumes is currently performed by one surface rendering or volume rendering approach. In this paper, we describe improvements developed in the field of volume rendering. We focused on three methods: parallelepiped face mapping: the rotation-projection method (with or without stereoscopy, with different matters and transparencies); the voxel ray-tracing method. We compared the possibilities of these different algorithms, in terms of quality of rendering, of computation load and as an essential aid to study the 3D organization of biological specimens.     

Three‐dimensional optical transfer function in differential confocal microscopy

To reveal the fundamental characteristics of differential confocal microscopy (DCM), its imaging properties were analysed by studying the 3D optical transfer function (OTF). The zero transfer at zero frequency along the axial direction in DCM, which has not been well understood and is considerably different from the transfer behaviour in conventional confocal microscopy (CM), was elucidated. The integral expressions of the OTFs for CM and DCM and the subsequent simulation results showed that DCMs have higher transfer capability than CM in the axial direction at medium and high frequencies. Conventionally, the relative optimal defocusing amount in DCMs are determined through calculations of the gradient of the point spread functions in the spatial domain. In contrast, in this study, the OTF performances were compared and the optimal defocusing amount was found to be between 5 and 7.     

Three‐dimensional analysis of injury conditions of single muscle fibers in small animals using phase‐contrast X‐ray imaging

Muscle damage can reduces the biological functions and lead to ultimately a disease state. For the reason, it is important to accurately check the state of an injury such as atrophy, and it is required to identify the state of fibers constituting the muscle. This study describes a novel method of analyzing single muscle fibers with injury conditions in three‐dimensions. The muscle fibers of the mice were visualized using phase‐contrast X‐ray projection the microstructure. In additions, it was possible to confirm the status by quantitatively analyzing the injury severity of muscle fibers. Significantly, the muscle conditions of multiple individuals were individually determined. This study could contributes to areas where it is very important to identify microdetailed and quantitative changes of state, such as new drug development.     

Three-dimensional visualization and physiologic evaluation of bile canaliculi in the rat liver slice by confocal laser scanning microscopy.

We evaluated the morphology and physiologic function of the bile canaliculi (BC) in the rat liver slice (RLS) by confocal laser scanning microscopy (CLSM). Lucifer yellow (LY) dye was injected into the RLS, and the distribution of LY was serially evaluated. After the injection of LY, hepatocytes were initially visualized, followed by visualization of the BC. There was no significant difference in the distribution of LY between zones 1 and 3 in the hepatic lobule. In zone 1, the reticular distribution of the BC was observed, whereas the part of BC was linearly visualized in zone 3 along the course of sinusoids. When changes in the bile canalicular fluorescence (BCF) were serially evaluated, the BCF was decreased to the minimal level (88% of the value obtained immediately after the LY injection) 10 min after the LY injection, and it tended to increase thereafter. The intralobular hepatocyte fluorescence (ILHF) was decreased to 58.9% of the initial value during the first 40 min. However, the ILHF was transiently increased 30 min after the LY injection, suggesting the possibility of reabsorption of LY by hepatocytes. Three-dimensional (3-D) reconstruction images of the BC facilitated the evaluation of the stereoscopic structure of BC. Confocal laser scanning microscopy facilitated the evaluation of structures and physiologic function of the BC.     

Multipoint scanning dual‐detection confocal microscopy for fast 3D volumetric measurement

We propose a multipoint scanning dual‐detection confocal microscopy (MS‐DDCM) system for fast 3D volumetric measurements. Unlike conventional confocal microscopy, MS‐DDCM can accomplish surface profiling without axial scanning. Also, to rapidly obtain 2D images, the MS‐DDCM employs a multipoint scanning technique, with a digital micromirror device used to produce arrays of effective pinholes, which are then scanned. The MS‐DDCM is composed of two CCDs: one collects the conjugate images and the other collects nonconjugate images. The ratio of the axial response curves, measured by the two detectors, provides a linear relationship between the height of the sample surface and the ratio of the intensity signals. Furthermore, the difference between the two images results in enhanced contrast. The normalising effect of the MS‐DDCM provides accurate sample heights, even when the reflectance distribution of the surface varies. Experimental results confirmed that the MS‐DDCM achieved high‐speed surface profiling with improved image contrast capability.     

体绘制技术在剂量三维可视化中的应用

本文基于光线投射方法的体绘制三维可视化用技术,在光线投射采样过程中,对阻光度分别基于CT值自动分类和手动自定义分类,基于感兴趣区进行自定义分类,对颜色值分量分别基于剂量值进行自动分类,基于处方剂量进行自定义分类.这些分类方式及不同组合,使医生可以根据临床需要对阻光度及颜色进行定义,从而使得剂量分布可以基于需要进行三维可视化显示,从而使医生能更直观地判断相关组织的剂量分布情况.此外,由于基于光线投射理论的体绘制技术无需构造中间几何图元,使得基于患者CT图像体数据的三维可视化细节更加清晰.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.