Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive‐index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high‐end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive‐index mismatch for high‐aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high‐angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point‐scanning confocal microscope and showed that a nearly index‐matched, micron‐sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.     

Direct Volume Rendering Methods for Cell Structures

The study of the complicated architecture of cell space structures is an important problem in biology and medical research. Optical cuts of cells produced by confocal microscopes enable two‐dimensional (2D) and three‐dimensional (3D) reconstructions of observed cells. This paper discuses new possibilities for direct volume rendering of these data. We often encounter 16 or more bit images in confocal microscopy of cells. Most of the information contained in these images is unsubstantial for the human vision. Therefore, it is necessary to use mathematical algorithms for visualization of such images. Present software tools as OpenGL or DirectX run quickly in graphic station with special graphic cards, run very unsatisfactory on PC without these cards and outputs are usually poor for real data. These tools are black boxes for a common user and make it impossible to correct and improve them. With the method proposed, more parameters of the environment can be set, making it possible to apply 3D filters to set the output image sharpness in relation to the noise. The quality of the output is incomparable to the earlier described methods and is worth increasing the computing time. We would like to offer mathematical methods of 3D scalar data visualization describing new algorithms that run on standard PCs very well. SCANNING 34: 367‐377, 2012. © 2012 Wiley Periodicals, Inc.     

Automated quantification and reconstruction of collagen matrix from 3D confocal datasets

The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered‐light confocal images of collagen gels. The computed quantitative data allowed us to create surface‐rendered 3D images of the investigated sample.     

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.     

Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.     

Three-dimensional visualization of insect morphology using confocal laser scanning microscopy

Cuticular structures of insects are often microscopic and intricately complex; among the most complex structures are male genitalia. Genitalic structures are essential in taxonomic and phylogenetic studies of insects. Using well‐described species from two disparate dipteran genera, we demonstrate the utility of confocal laser scanning microscopy for studying the morphological characters of fly genitalia by taking advantage of the autofluorescent properties of cuticle material. Reconstructions of confocal data sets obtained from genitalic structures embedded in two commonly used entomological mounting media (euparal and glycerin jelly) are presented. Aberration artefacts often observed in confocal data obtained from thick specimens were analysed and strategies for their minimization are discussed. Our results indicate that confocal laser scanning microscopy and 3D reconstruction are excellent techniques for visualizing small, complex, autofluorescent structures in flies. These techniques could have a profound impact on the quality of information provided by 3D representations of insect structures over more traditional methods of visualization.     

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 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.     

Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3-D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3-D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two-dimensional (2-D) DIC images allow visualization of the same population of collagen fibrils as observed in 2-D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time-lapse 2-D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3-D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell-matrix mechanical interactions will likely require a combination of both wide-field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell-induced compaction and alignment of ECM which occur over a longer time course.     

An automatic method for identifying appropriate gradient magnitude for 3D boundary detection of confocal image stacks

Gradients play an important role in 2D image processing. Many edge detection algorithms are gradient‐based. We are interested in 3D boundary detection which can be considered as an extension of 2D edge detection in 3D space. In this paper, an algorithm to automatically and quantitatively measure the suitability of gradient magnitudes in detection of 3D boundary points of confocal image stacks is presented. A Measurement Function is defined to evaluate the suitability of each gradient magnitude chosen to be the threshold for 3D boundary detection. The application of Gauss's Divergence Theorem provides a solution to calculate the Measurement Function numerically. The gradient magnitude at which the maximum of the Measurement Function is achieved can be utilized as the most appropriate threshold for gradient‐based boundary detection and other operations like volume visualization.     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

Micro‐CT versus synchrotron radiation phase contrast imaging of human cochlea

High‐resolution images of the cochlea are used to develop atlases to extract anatomical features from low‐resolution clinical computed tomography (CT) images. We compare visualization and contrast of conventional absorption‐based micro‐CT to synchrotron radiation phase contrast imaging (SR‐PCI) images of whole unstained, nondecalcified human cochleae. Three cadaveric cochleae were imaged using SR‐PCI and micro‐CT. Images were visually compared and contrast‐to‐noise ratios (CNRs) were computed from n = 27 regions‐of‐interest (enclosing soft tissue) for quantitative comparisons. Three‐dimensional (3D) models of cochlear internal structures were constructed from SR‐PCI images using a semiautomatic segmentation method. SR‐PCI images provided superior visualization of soft tissue microstructures over conventional micro‐CT images. CNR improved from 7.5 ± 2.5 in micro‐CT images to 18.0 ± 4.3 in SR‐PCI images (p < 0.0001). The semiautomatic segmentations yielded accurate reconstructions of 3D models of the intracochlear anatomy. The improved visualization, contrast and modelling achieved using SR‐PCI images are very promising for developing atlas‐based segmentation methods for postoperative evaluation of cochlear implant surgery.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

Usefulness of hard X‐ray microscope using synchrotron radiation for the structure analysis of insects

Three‐dimensional (3D) printing technology has the advantage of enabling specific visualization of creative ideas. Since synchrotron based images can provide high sensitivity and high resolution, they are a very useful technology to identify the 3D anatomy of microscale samples. X‐ray images using such synchrotron radiation are grafted to 3D printing technology. We can be obtained 3D images and modeling data of an ant using synchrotron radiation, and then, it were outputted with the 3D printer. A new way to identify the usefulness of the structure analysis is then found by visualizing the micro‐internal structure of diverse biomedical samples and creating an enlarged model. This study suggests methods of utilizing a 3D printed model produced through synchrotron radiation imaging in various fields such as bioengineering, medical, and education.     

ScatterJ: An ImageJ plugin for the evaluation of analytical microscopy datasets

We present ScatterJ, an ImageJ plugin that allows for extracting qualitative as well as quantitative information from analytical microscopy datasets. A large variety of analytical microscopy methods are used to obtain spatially resolved chemical information. The resulting datasets are often large and complex, and can contain information that is not obvious or directly accessible. ScatterJ extends and complements existing methods to extract information on correlation and colocalization from pairs of species‐specific or element‐specific maps. We demonstrate the possibilities to extract information using example datasets from biogeochemical studies, although the plugin is not restricted to this type of research. The information that we could extract from our existing data helped to further our understanding of biogeochemical processes such as mineral formation or heavy metal sorption. ScatterJ can be used for a variety of different two‐dimensional (2D) and three‐dimensional (3D) datasets such as energy‐dispersive X‐ray spectroscopy maps, 3D confocal laser scanning microscopy maps, and 2D scanning transmission X‐ray microscopy maps.     

Three-dimensional volume reconstruction of extracellular matrix proteins in uveal melanoma from fluorescent confocal laser scanning microscope images

The distribution of looping patterns of laminin in uveal melanomas and other tumours has been associated with adverse outcome. Moreover, these patterns are generated by highly invasive tumour cells through the process of vasculogenic mimicry and are not therefore blood vessels. Nevertheless, these extravascular matrix patterns conduct plasma. The three‐dimensional (3D) configuration of these laminin‐rich patterns compared with blood vessels has been the subject of speculation and intensive investigation. We have developed a method for the 3D reconstruction of volume for these extravascular matrix proteins from serial paraffin sections cut at 4 µm thicknesses and stained with a fluorescently labelled antibody to laminin ( Maniotis et al., 2002 ). Each section was examined via confocal laser‐scanning focal microscopy (CLSM) and 13 images were recorded in the Z‐dimension for each slide. The input CLSM imagery is composed of a set of 3D subvolumes (stacks of 2D images) acquired at multiple confocal depths, from a sequence of consecutive slides. Steps for automated reconstruction included (1) unsupervised methods for selecting an image frame from a subvolume based on entropy and contrast criteria, (2) a fully automated registration technique for image alignment and (3) an improved histogram equalization method that compensates for spatially varying image intensities in CLSM imagery due to photo‐bleaching. We compared image alignment accuracy of a fully automated method with registration accuracy achieved by human subjects using a manual method. Automated 3D volume reconstruction was found to provide significant improvement in accuracy, consistency of results and performance time for CLSM images acquired from serial paraffin sections.     

Modelling of three-dimensional fluorescence images of muscle fibres: an application of the three-dimensional optical transfer function

Striated muscle fibres can be modelled by a simple geometry, which has allowed three-dimensional (3-D) images in conventional and confocal microscopes to be calculated. This model is useful for comparing different imaging methods and represents a simple example of an application of the 3-D optical transfer function (OTF) for the system. The rejection of out-of-focus blur is demonstrated, and the effects of fibre thickness and confocal pinhole size on image contrast are investigated. The effects of using a simple filter for image enhancement are studied, elucidating the characteristics of the OTF.     

A new method of imaging particle tracks in solid state nuclear track detectors

Solid state nuclear track detectors are used to determine the concentration of α particles in the environment. The standard method for assessing exposed detectors involves 2D image analysis. However 3D imaging has the potential to provide additional information relating to angle as well as to differentiate clustered hit sequences and possibly energy of α particles but this could be time consuming. Here we describe a new method for rapid high-resolution 3D imaging of solid state nuclear track detectors. A 'LEXT' OLS3100 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) was used in confocal mode to successfully obtain 3D image data on four CR-39 plastic detectors. Three-dimensional visualization and image analysis enabled characterization of track features. This method may provide a means of rapid and detailed 3D analysis of solid state nuclear track detectors.     

Attenuation correction in confocal laser microscopes: a novel two-view approach

Confocal microscopy is a three‐dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth‐dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal‐to‐noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two‐view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180° with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model‐based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space‐filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth‐dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two‐view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.