The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector ...for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.
Measurement and analysis of bone morphometry in 3D micro-computed tomography volumes using automated image processing and analysis improve the accuracy, consistency, reproducibility, and speed of ...preclinical osteological research studies. Automating segmentation and separation of individual bones in 3D microcomputed tomography volumes of murine models presents significant challenges considering partial volume effects and joints with thin spacing, i.e., 50 to 100 μm. In this paper, novel hybrid splitting filters are presented to overcome the challenge of automated bone separation. This is achieved by enhancing joint contrast using rotationally invariant second-derivative operators. These filters generate split components that seed marker-controlled watershed segmentation. In addition, these filters can be used to separate metaphysis and epiphysis in long bones, e.g., femur, and remove the metaphyseal growth plate from the detected bone mask in morphometric measurements. Moreover, for slice-by-slice stereological measurements of long bones, particularly curved bones, such as tibia, the accuracy of the analysis can be improved if the planar measurements are guided to follow the longitudinal direction of the bone. In this paper, an approach is presented for characterizing the bone medial axis using morphological thinning and centerline operations. Building upon the medial axis, a novel framework is presented to automatically guide stereological measurements of long bones and enhance measurement accuracy and consistency. These image processing and analysis approaches are combined in an automated streamlined software workflow and applied to a range of in vivo micro-computed tomography studies for validation.
Iterative tomographic reconstruction has been established as a viable alternative for data analysis in phase-sensitive x-ray imaging based on the edge-illumination principle. However, previously ...published approaches did not account for drifts of optical elements during a scan, which can lead to artefacts. Up to now, the strategy to reduce these artefacts was to acquire additional intermediate flat field images, which were used to correct the sinograms. Here, we expand the theoretical model to take these effects into account and demonstrate a significant reduction of (ring)-artefacts in the final reconstructions, while allowing for a significant reduction of scan time and dose. We further improve the model by including the capability to reconstruct combined absorption and phase contrast slices, which we experimentally demonstrate to deliver improved contrast to noise ratios compared to previously employed single shot approaches.
A multimodal micro-computed tomography (CT) and multi-spectral structured light imaging (SLI) system is introduced and systematically analyzed to test its feasibility to aid in margin delineation ...during breast conserving surgery (BCS). Phantom analysis of the micro-CT yielded a signal-to-noise ratio of 34, a contrast of 1.64, and a minimum detectable resolution of 240 μm for a 1.2 min scan. The SLI system, spanning wavelengths 490 nm to 800 nm and spatial frequencies up to 1.37 mm−1, was evaluated with aqueous tissue simulating phantoms having variations in particle size distribution, scatter density, and blood volume fraction. The reduced scattering coefficient, μs′ and phase function parameter, γ, were accurately recovered over all wavelengths independent of blood volume fractions from 0% to 4%, assuming a flat sample geometry perpendicular to the imaging plane. The resolution of the optical system was tested with a step phantom, from which the modulation transfer function was calculated yielding a maximum resolution of 3.78 cycles per mm. The three dimensional spatial co-registration between the CT and optical imaging space was tested and shown to be accurate within 0.7 mm. A freshly resected breast specimen, with lobular carcinoma, fibrocystic disease, and adipose, was imaged with the system. The micro-CT provided visualization of the tumor mass and its spiculations, and SLI yielded superficial quantification of light scattering parameters for the malignant and benign tissue types. These results appear to be the first demonstration of SLI combined with standard medical tomography for imaging excised tumor specimens. While further investigations are needed to determine and test the spectral, spatial, and CT features required to classify tissue, this study demonstrates the ability of multimodal CT/SLI to quantify, visualize, and spatially navigate breast tumor specimens, which could potentially aid in the assessment of tumor margin status during BCS.
Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to ...monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location. Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.
Compared to standard tomographic reconstruction, iterative approaches offer the possibility to account for extraneous experimental influences, which allows for a suppression of related artifacts. ...However, the inclusion of corresponding parameters in the iterative forward model typically leads to longer computation times. Here, we demonstrate experimentally for phase sensitive X-ray imaging based on the edge illumination principle that inadequately sampled illumination curves result in ring artifacts in tomographic reconstructions. We take advantage of appropriately sampled illumination curves instead, which enables us to eliminate the corresponding parameter from the forward model and substantially increase computational speed. In addition, we demonstrate a 30\% improvement in spatial resolution of the iterative approach compared with the standard non-iterative single shot approach. Further, we report on several significant improvements in our numerical implementation of the iterative approach, which we make available online with this publication. Finally, we show that the combination of both experimental and algorithmic advancement lead to a total speed increase by one order of magnitude and an improved contrast to noise ratio in the reconstructions.
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