MicroCT for developmental biology: A versatile tool for high‐contrast 3D imaging at histological resolutions

Understanding developmental processes requires accurate visualization and parameterization of three‐dimensional embryos. Tomographic imaging methods offer automatically aligned and calibrated volumetric images, but the usefulness of X‐ray CT imaging for developmental biology has been limited by the low inherent contrast of embryonic tissues. Here, I demonstrate simple staining methods that allow high‐contrast imaging of embryonic tissues at histological resolutions using a commercial microCT system. Quantitative comparisons of images of chick embryos treated with different contrast agents show that three very simple methods using inorganic iodine and phosphotungstic acid produce overall contrast and differential tissue contrast for X‐ray imaging at least as high as that obtained with osmium. The stains can be used after any common fixation and after storage in aqueous or alcoholic media, and on a wide variety of species. These methods establish microCT imaging as a useful tool for comparative developmental studies, embryo phenotyping, and quantitative modeling of development. Developmental Dynamics 238:632–640, 2009. © 2009 Wiley‐Liss, Inc.     

Micro‐Computed Tomography for Visualizing Limb Skeletal Regeneration in Young Xenopus Frogs

For studies of vertebrate limb regeneration it is often desirable to visualize the regenerated skeleton, which is mostly cartilage, and also section the specimen for histological or immunohistochemical visualization of other tissues. However, the normal skeletal staining techniques are incompatible with immunohistochemistry. Here, we describe a contrast‐based micro‐computed tomography (microCT) method for direct and nondestructive observation of regenerated cartilage spikes in Xenopus frog limbs. In addition, we show that contrast based microCT imaging is compatible with immunohistochemistry protocols. This approach provides versatile and high contrast images of the cartilage allowing us to measure the regenerated skeletal structure in detail as well as carrying out the other types of analysis. It opens a wide range of potential microCT applications in research on vertebrate limb regeneration. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Segmentation techniques for analysis of bone by three-dimensional computed tomographic imaging.

Improved methods for evaluation and quantification of the three-dimensional (3D) architecture of bone are needed in order to more fully understand the role of trabecular architecture in bone strength. Computed tomography (microCT) is capable of examining bone at resolutions below 30 microm (isotropic), with collection of a three-dimensional data set which can then be subjected to image analysis. In this paper, we discuss automated methods for important steps in this analysis, including methods for (1) segmenting the image into bone and background; (2) defining the volume of interest for determination of structural parameters; and (3) segmenting the bone into trabecular and cortical components. Evaluation of bone structure using these techniques provides new information about the 3D architecture of bone tissue, and may be useful for evaluation of structural changes in bone caused by aging, disease, or drug treatment.     

TLD assessment of mouse dosimetry during microCT imaging

Advances in laboratory animal imaging have provided new resources for noninvasive biomedical research. Among these technologies is microcomputed tomography (microCT) which is widely used to obtain high resolution anatomic images of small animals. Because microCT utilizes ionizing radiation for image formation, radiation exposure during imaging is a concern. The objective of this study was to quantify the radiation dose delivered during a standard microCT scan. Radiation dose was measured using thermoluminescent dosimeters (TLDs), which were irradiated employing an 80 kVp x-ray source, with 0.5 mm A1 filtration and a total of 54 mA s for a full 360 deg rotation of the unit. The TLD data were validated using a 3.2 cm3 CT ion chamber probe. TLD results showed a single microCT scan air kerma of 78.0 +/- 5.0 mGy when using a poly(methylmethacrylate) (PMMA) anesthesia support module and an air kerma of 92.0 +/- 6.0 mGy without the use of the anesthesia module. The validation CT ion chamber study provided a measured radiation air kerma of 81.0 +/- 4.0 mGy and 97.0 +/- 5.0 mGy with and without the PMMA anesthesia module, respectively. Internal TLD analysis demonstrated an average mouse organ radiation absorbed dose of 76.0 +/- 5.0 mGy. The author's results have defined x-ray exposure for a routine microCT study which must be taken into consideration when performing serial molecular imaging studies involving the microCT imaging modality.     

Three-dimensional visualization of bioactive glass-bone integration in a rabbit tibia model using synchrotron X-ray microcomputed tomography

Synchrotron X-ray microcomputed tomography (SR microCT), with a micron resolution, was used to evaluate the osteoconduction and osteointegration by borate bioactive glass after implantation 12 weeks in a rabbit tibia model. The study focused on the biomaterial-bone interface. Results from SR microCT two-dimensional and three-dimensional (3D) reconstructions provided precise imaging of the biomaterial-bone integration and detailed microarchitecture of both the bone-like glass graft and the newly formed trabecular bone. Osteoconduction, the formation of new trabecular bone within a tibia defect, occurred only in the tibiae implanted with teicoplanin-loaded borate glass but not in those with teicoplanin-loaded CaSO(4) beads, indicating the excellent biocompatibility of the glass implants. 3D reconstruction of the tibiae also showed the infiltration of vascular tissue in both the bioactive glass graft and the new trabecular bone. This study indicates that SR microCT can serve as a valuable complementary technique for imaging bone repair when using bioactive glass implants.     

In vivo quantification of subcutaneous and visceral adiposity by micro-computed tomography in a small animal model

Accurate and precise techniques that identify the quantity and distribution of adipose tissue in vivo are critical for investigations of adipose development, obesity, or diabetes. Here, we tested whether in vivo micro-computed tomography (microCT) can be used to provide information on the distribution of total, subcutaneous and visceral fat volume in the mouse. Ninety C57BL/6J mice (weight range: 15.7-46.5 g) were microCT scanned in vivo at 5 months of age and subsequently sacrificed. Whole body fat volume (base of skull to distal tibia) derived from in vivo microCT was significantly (p<0.001) correlated with the ex vivo tissue weight of discrete perigonadal (R(2)=0.94), and subcutaneous (R(2)=0.91) fat pads. Restricting the analysis of tissue composition to the abdominal mid-section between L1 and L5 lumbar vertebrae did not alter the correlations between total adiposity and explanted fat pad weight. Segmentation allowed for the precise discrimination between visceral and subcutaneous fat as well as the quantification of adipose tissue within specific anatomical regions. Both the correlations between visceral fat pad weight and microCT determined visceral fat volume (R(2)=0.95, p<0.001) as well as subcutaneous fat pad weight and microCT determined subcutaneous fat volume (R(2)=0.91, p<0.001) were excellent. Data from these studies establish in vivo microCT as a non-invasive, quantitative tool that can provide an in vivo surrogate measure of total, visceral, and subcutaneous adiposity during longitudinal studies. Compared to current imaging techniques with similar capabilities, such as microMRI or the combination of DEXA with NMR, it may also be more cost-effective and offer higher spatial resolutions.     

A synchrotron radiation microtomography system for the analysis of trabecular bone samples.

X-ray computed microtomography is particularly well suited for studying trabecular bone architecture, which requires three-dimensional (3-D) images with high spatial resolution. For this purpose, we describe a three-dimensional computed microtomography (microCT) system using synchrotron radiation, developed at ESRF. Since synchrotron radiation provides a monochromatic and high photon flux x-ray beam, it allows high resolution and a high signal-to-noise ratio imaging. The principle of the system is based on truly three-dimensional parallel tomographic acquisition. It uses a two-dimensional (2-D) CCD-based detector to record 2-D radiographs of the transmitted beam through the sample under different angles of view. The 3-D tomographic reconstruction, performed by an exact 3-D filtered backprojection algorithm, yields 3-D images with cubic voxels. The spatial resolution of the detector was experimentally measured. For the application to bone investigation, the voxel size was set to 6.65 microm, and the experimental spatial resolution was found to be 11 microm. The reconstructed linear attenuation coefficient was calibrated from hydroxyapatite phantoms. Image processing tools are being developed to extract structural parameters quantifying trabecular bone architecture from the 3-D microCT images. First results on human trabecular bone samples are presented.     

Novel approach for quantification of porosity for biomaterial implants using microcomputed tomography (microCT)

Porous bioceramics have been widely investigated in orthopaedic tissue engineering. Attention has been given to manufacturing of a porous bioceramic that mimics the trabecular bone structure for proper bone regeneration. With the advance of biomedical imaging through microcomputed tomography (microCT), this study attempted to quantify the pore structure of different bioceramics. Two bioceramic blocks (BSC and ChronOS) were synthesized by two methods. The specification claimed the porosity of the bioceramic ranged from 40% to 70%. Six blocks of each bioceramic were evaluated by conventional water immersion method and microCT. The pore size and connectivity were evaluated with standardized protocols. By the water immersion method, the porosity of BSC and ChronOS was 60.4% and 74.7%, respectively. The three-dimensional results of microCT showed that BSC porosity was 26.2% and ChronOS was 60.0%. The pore connectivity was evaluated to be 2.6 for BSC and 39.7 for ChronOS. ChronOS had functional pores with 200 microm to 400 microm in diameter (87.8%+/-0.5%), which is significantly more than 52.8%+/-11.5% of pores in BSC (p<0.05). Providing information on the functional pores objectively, the microCT evaluation serves as a good standard for specification of the bioceramic-related implants.     

Correlation of energy dispersive diffraction signatures and microCT of small breast tissue samples with pathological analysis

Identification of specific tissue types in conventional mammographic examinations is extremely limited. However, the use of x-ray diffraction effects during imaging has the potential to characterize the tissue types present due to the fact that each tissue type produces its own unique diffraction signature. Nevertheless, the analysis and categorization of these diffraction signatures by tissue type can be hampered by the inhomogeneous nature of breast tissue, leading to categorization errors where several types are present. This work aims to reduce sample categorization errors by combining spectral diffraction signature collection with sample imaging, giving more detailed data on the composition of each sample. Diffraction microCT was carried out on 19 unfixed breast tissue samples using an energy resolving translate-rotate CT system. High-resolution transmission microCT images were also recorded for comparison and sample composition analysis. Following imaging, the samples were subjected to histopathological analysis. Reconstructing on various momentum transfer regions allows different tissue types to be identified in the diffraction images. Results show a correlation between measured x-ray diffraction images and stained histopathological tissue sections. X-ray diffraction signatures generated from the measured data were categorized and analysed, with a t-test indicating that they have the potential for use in tissue type identification.     

The Quantitative Structural and Compositional Analyses of Degenerating Intervertebral Discs Using Magnetic Resonance Imaging and Contrast-Enhanced Micro-Computed Tomography

The intervertebral disc (IVD) is susceptible to degenerative changes that are associated with low back pain. Murine models are often used to investigate the mechanistic changes in the development, aging, and diseased states of the IVD, yet the detection of early degenerative changes in structure is challenging because of the minute size of the murine IVDs. Histology is the gold standard for examining the IVD structure, but it is susceptible to sectioning artifacts, spatial biases, and requires the destructive preparation of the sample. We have previously demonstrated the feasibility of using Ioversol for the contrast-enhanced micro-computed tomography (microCT) to visualize and quantitate the intact healthy murine IVD. In this work, we demonstrate utility of this approach to monitor the longitudinal changes of in vitro nucleolytic- and mechanical injury- degeneration models of the murine discs and introduce novel quantitative metrics to characterize the structure and composition of the IVD. Moreover, we compared the imaging quality and quantitation of these in vitro models to magnetic resonance imaging (MRI) and histology. Stab puncture, trypsin injection, and collagenase injection all induced detectable and significant changes in structure and composition of the discs overtime. Compared to MRI and histology, contrast-enhanced microCT produced superior images that capture the degenerative progression in these models. Contrast-enhanced microCT was also capable of monitoring the structural deteriorations via the changes in disc height and volume, and novel the nucleus pulposus intensity/disc intensity (NI/DI) parameter provides a surrogate measure of proteoglycan composition (R = 0.96). Overall, this approach allows for the nondestructive monitoring of the structure and composition of the IVD at very high resolutions.     

Kardiopulmonales Gefäßsystem

In recent years microcomputed tomography (microCT) has become more and more important in basic research. Now commercial microCT scanners are available. Thus, it is very likely that this new, accurate and promising method for three-dimensional and non-destructive quantitative evaluation of intact tissues including vessels will be applied more frequently. The review provides a survey of the basic technology of microCT and its current use for high resolution three-dimensional morphometric and functional analysis within the cardio-pulmonary vascular system.     

Estimating structural properties of trabecular bone from gray-level low-resolution images

In this paper the relationship between three-dimensional histomorphometric parameters derived from microCT and MRI images of distal radius trabecular bone samples is studied. microFE analysis of the trabecular samples is performed and Young's modulus for cranio-caudal direction is calculated. Most of the MRI and microCT parameters correlate significantly with, respectively, MRI and microCT estimates of bone volume fraction. For some of the parameters strong correlation between microCT and MRI results is also observed. However, in these cases there simultaneously exists correlation between: microCT parameter and microCT bone volume fraction; microCT and MRI bone volume fraction; MRI bone volume fraction and MRI parameter. It is found that, comparing to bone volume fraction, histomorphometric information derived from binarized MRI images does not improve estimation of the Young's modulus of trabecular bone samples (calculated for "gold standard" microCT data). Thus a novel method of "optimal paths" analysis of gray-level MRI images is introduced. "Optimal paths" parameters improve estimation of the Young's modulus of trabecular bone samples. They also provide surrogate, gray-level image-based measure of trabecular thickness.     

REST-PEEP: reduced scan time phase-encoded echo planar imaging

Echo planar methodology can be used for rapid acquisition in three dimensions of k-space. There are two groups of sequences which have thus far been implemented. The first group is characterized by three-dimensional acquisition from a single RF excitation. Echo planar shift mapping and echo volume imaging are single-shot chemical shift imaging and three-dimensional spatial imaging techniques, respectively. Even though these methods are extremely fast, their spatial and spectral resolutions are poor. In the second group of sequences, echo planar imaging acquisition is repeated for every phase-encode gradient step to improve upon the spatial resolution and signal-to-noise ratio at the expense of acquisition time. Here we report a novel combination of these two groups of sequences aimed at providing additional flexibility in trade-offs between the spectral bandwidth, signal-to-noise ratio, spatial resolution and imaging time. Preliminary results for both chemical shift imaging and three-dimensional spatial imaging are presented. The chemical shift imaging was optimized for excitation of the N-acetyl aspartate (NAA). An interesting consequence of our approach is that the Nyquist ghost is transferred from the spectrum to the metabolite image.     

Postcranial skeletal pneumaticity: a case study in the use of quantitative microCT to assess vertebral structure in birds

Limb elements in birds have been characterized as exhibiting a reduction in trabecular bone, thinner cortices and decreased bending strength when pneumatized, yet it is unclear if these characteristics generalize to the axial skeleton. Thin section techniques, the traditional gold standard for bone structure studies, have most commonly been applied to the study of avian bone. This destructive technique, however, makes it subsequently impossible to use the same samples in experimental testing systems that allow researchers to correlate structure with the mechanical properties of the bone. Micro-computed tomography (microCT), a non-destructive X-ray imaging technique, can be used to assess the effect of pneumatization on vertebral cortical and trabecular bone through virtual extraction and structural quantification of each tissue type. We conducted a preliminary investigation of the application of microCT methods to the study of cortical and trabecular bone structure in a small sample of pneumatic and apneumatic thoracic vertebrae. The sample consisted of two similar-sized anatids, Aix sponsa (n = 7) and Oxyura jamaicensis (n = 5). Volumes of interest were created that contoured (outlined) the boundaries of the ventral cortical bone shell, the trabecular compartment and the whole centrum (cortical bone + trabecular bone), and allowed independent structural analysis of each volume of interest. Results indicated that bone volume fraction of the whole centrum was significantly higher in the apneumatic O. jamaicensis than in the pneumatized A. sponsa (A. sponsa = 36%, O. jamaicensis = 48%, P < 0.05). In contrast, trabecular bone volume fraction was similar between the two species. The ventral cortical bone shell was approximately 23% thinner (P < 0.05) in A. sponsa (0.133 mm) compared with apneumatic O. jamaicensis (0.172 mm). This case study demonstrates that microCT is a powerful non-destructive imaging technique that may be applied to the three-dimensional study of avian bone. The preliminary results suggest that pneumatic and apneumatic vertebrae of comparably sized avian species differ in relative bone volume, with the largest difference apparent at the level of the cortex, and not within trabecular bone. The presence of relatively thin cortices in pneumatic vertebrae is consistent with previous studies contrasting diaphyseal cortical bone between pneumatic and apneumatic long bones. Methodological issues related to this and any comparative microCT study of bone structure are discussed.     

In vivo bone regeneration with injectable calcium phosphate biomaterial: a three-dimensional micro-computed tomographic, biomechanical and SEM study

This in vivo study investigated the efficiency of an injectable calcium phosphate bone substitute (IBS) for bone regenerative procedures through non-destructive three-dimensional (3D) micro-tomographic (microCT) imaging, biomechanical testing with a non-destructive micro-indentation technique and 2D scanning electron microscopy (SEM) analysis. The injectable biomaterial was obtained by mixing a biphasic calcium phosphate (BCP) ceramic mineral phase and a cellulosic polymer. The BCP particles were 200-500 microm or 80-200 microm in diameter. The injectable material was implanted for 6 weeks into critical-sized bone defects at the distal end of rabbit femurs. Extensive new bone apposition was noted with both 2D and 3D techniques. Micro-CT showed that newly formed bone was in perfect continuity with the trabecular host bone structure and demonstrated the high interconnectivity of the restored bone network. For both IBS formulations, SEM and microCT gave very close measurements. The only detected significant difference concerned the amount of newly formed bone obtained with IBS 80-200 that appeared significantly higher with microCT analysis than with SEM (p=0.00007). Student t-tests did not show any significant difference in the amount of newly formed bone and remaining ceramic obtained from microCT analysis or SEM. Regression analysis showed satisfactory correlation between both the amount of newly formed bone and remaining ceramic obtained from microCT or SEM. For IBS 200-500, the newly formed bone rate inside the defect was 28.0+/-5.2% with SEM and yield strength of the samples was 18.8+/-5.4 MPa. For IBS 80-200, the newly formed bone rate inside the defect was 31.7+/-5.1% with SEM and yield strength of the samples was 26.8+/-4.5 MPa. Yield strength appeared well correlated with the amount of newly formed bone, specially observed with microCT. This study showed the ability of non-destructive techniques to investigate biological and mechanical aspects of bone replacement with injectable biomaterials.     

A method of image registration for small animal, multi-modality imaging

Many research institutions have a full suite of preclinical tomographic scanners to answer biomedical questions in vivo. Routine multi-modality imaging requires robust registration of images generated by various tomographs. We have implemented a hardware registration method for preclinical imaging that is similar to that used in the combined positron emission tomography (PET)/computed tomography (CT) scanners in the clinic. We designed an imaging chamber which can be rigidly and reproducibly mounted on separate microPET and microCT scanners. We have also designed a three-dimensional grid phantom with 1288 lines that is used to generate the spatial transformation matrix from software registration using a 15-parameter perspective model. The imaging chamber works in combination with the registration phantom synergistically to achieve the image registration goal. We verified that the average registration error between two imaging modalities is 0.335 mm using an in vivo mouse bone scan. This paper also estimates the impact of image misalignment on PET quantitation using attenuation corrections generated from misregistered images. Our technique is expected to produce PET quantitation errors of less than 5%. The methods presented are robust and appropriate for routine use in high throughput animal imaging facilities.     

Nondestructive micro-computed tomography for biological imaging and quantification of scaffold-bone interaction in vivo

Scaffolds, also called bioscaffolds, are needed in all tissue engineering applications as carriers for cells and biochemical factors, as constructs providing appropriate mechanical conditions, or as a combination of the two. The aim of this paper is to present recent developments in micro-computed tomography (microCT) analyses of scaffolds. The focus will be on imaging and quantification aspects in bone research, and will deal with the assessment of scaffold architecture and how it interacts with bone tissue. We show that micro-architectural imaging is a nondestructive and noninvasive procedure that allows a precise three-dimensional (3D) measurement of scaffold architecture. Direct microCT-based image analysis allows to accurately quantify scaffold porosity, surface area, and 3D measures such as pore size, pore distribution, and strut thickness; furthermore, it allows for a precise measurement of bone growth into the scaffold and onto its surface. This methodology is useful for quality control of scaffold fabrication processes, to assess scaffold degradation kinetics, and to assess bone tissue response. Even more so, in combination with bioreactors or in vivo animal models, microCT allows to qualitatively and quantitatively assess the spatial and temporal mineralization of bone tissue formation in scaffolds; such longitudinal studies improve the assessment of bone response due to scaffold architecture. Computational models will be helpful in further analyses of these data in order to improve our understanding of mechanical and biochemical stimuli on bone formation, and are likely to provide valuable knowledge to optimize scaffold design.     

A microPET/CT system for in vivo small animal imaging

A microCT scanner was designed, fabricated and integrated with a previously reported microPET II scanner (Tai et al 2003 Phys. Med. Biol. 48 1519, Yang et al 2004 Phys. Med. Biol. 49 2527), forming a dual modality system for in vivo anatomic and molecular imaging of the mouse. The system was designed to achieve high-spatial-resolution and high-sensitivity PET images with adequate CT image quality for anatomic localization and attenuation correction with low x-ray dose. The system also has relatively high throughput for screening, and a flexible gantry and user interface. X-rays were produced by a 50 kVp, 1.5 mA fixed tungsten anode tube, with a focal spot size of 70 microm. The detector was a 5 x 5 cm(2) photodiode detector incorporating 48 microm pixels on a CMOS array and a fast gadolinium oxysulfide (GOS) intensifying screen. The microCT system has a flexible C-arm gantry design with adjustable detector positioning, which acquires CT projection images around the common microPET/CT bed. The design and the initial characterization of the microCT system is described, and images of the first mouse scans with microPET/CT scanning protocols are shown.     

How does scanner choice and 3D model resolution affect data accuracy?

Researchers using digital methods often collect data from 3D models at different resolutions, obtained using different scanning techniques. Although previous research has sought to understand whether scanning method and model resolution affect data accuracy, no study has systematically evaluated the sources of error associated with scanning method, data acquisition method and model resolution with the aim of providing practical recommendations about the model resolution required to yield sufficiently accurate data for specimens of given sizes. In this study, using data taken from primate specimens of three broad size categories, we test whether 3D models obtained using five different scanners (Breuckmann SmartSCAN, DAVID/HP 3D Pro S3, NextEngine 2020i, Creaform Go!Scan 20 and microCT/clinicalCT) yield accurate measurements. We assess whether caliper measurements can be used alongside measurements collected from 3D surface models, whether scanning resolution affects measurement accuracy, and how scan resolution, estimated using each scanner's proprietary software, compares to model resolution measured in a standardized way. Each scanner produces 3D models that yield accurate measurements for each size category, however, combining caliper data with those taken from digital models can be problematic. Our results indicate that the accuracy of measurements taken from 3D models depends on both object size and model resolution. Based on our findings, we recommend that small specimens should be scanned at <0.3 mm, medium specimens at 0.3–0.7 mm, and large specimens at 0.3–0.5 mm resolutions if data taken from 3D surface models are to be combined with caliper datasets. We further show, for the first time, that discrepancies in estimated final model resolution are frequently observed across software packages. We therefore recommend that researchers ensure that final model resolutions are adequate based on specimen size and are independently verified using a software package other than the scanner's proprietary software. Finally, we consider the implications of the findings that measurements obtained from surface models are variably consistent with those obtained using calipers.     

Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs

Multiphoton microscopy of cleared tissue has previously been demonstrated to generate large three-dimensional (3D) volumetric image data on entire intact mouse organs using intrinsic tissue fluorescence. This technique holds great promise for performing 3D virtual biopsies, providing unique information on tissue morphology, and guidance for subsequent traditional slicing and staining. Here, we demonstrate the use of fluorescence lifetime imaging in cleared organs for achieving molecular contrast that can reveal morphologically distinct structures, even in the absence of knowledge of the underlying molecular source. In addition, we demonstrate the power of multimodal imaging, combining multiphoton fluorescence, second harmonic generation, and lifetime imaging to reveal exceptional morphological detail in an optically cleared mouse knee.