Hybrid imaging: combining fluorescence molecular tomography with magnetic resonance imaging

Optical imaging techniques, in particular fluorescence imaging is nowadays widely used in biomedical research. Among those optical imaging approaches, fluorescence microscopy is one of the most established and routinely used techniques in daily research. Unfortunately microscopy techniques are only applicable to study molecular and cellular processes in cell culture or tissue samples. Therefore an imaging strategy which would allow to apply the wealth of tools that have been developed for optical microscopy to intact organisms such as mice would be very attractive. Fluorescence molecular tomography (FMT) is such a promising and powerful macroscopic imaging technique. Due to significant technological progress during the last years FMT has emerged as interesting molecular imaging solution for various fields in biomedical research. FMT allows a three-dimensional reconstruction of fluorophore distribution in vivo by scanning an excitation source pattern on the sample surface and collecting the diffuse light by an array detector. In contrast to first contact setup implementations where the animal is place vertically in a liquid filled imaging chamber connected to a limited number of source and detector fibers, recent setups provide a more convenient horizontal non-contact measurement geometry. These setups are more flexible and have a large number of source/detector pairs which leads to improved reconstructed image resolution. Compared to straightforward implementations of planar imaging techniques where quantitative measurements are impossible to achieve, FMT allows depth resolved information and therefore the quantification of fluorophore distributions which is important for biological investigations. The major advantage of optical imaging techniques are the high sensitivity which makes it suitable to detect low concentration of target molecules in tissue. Furthermore, optical reporter systems such as fluorescent dyes or bioluminescent and fluorescent reporter proteins are stable molecules and can be easily targeted to report on specific molecular processes in vivo. Additionally, the techniques are relatively inexpensive, which facilitates their protrusion into the biomedical research community. The development and construction of a non-contact FMT setup was the first goal of this thesis. In a preliminary step the theoretical background of FMT and especially the concepts allowing for non-contact FMT were discussed in details. A second section describes the technical realization of the setup including further improvements compared to already published examples such as an integrated filter switch mechanism and preparative steps for multispectral imaging. Different in vivo applications demonstrated a proper function of the implemented method: In a first experiment load induced changes in bone osteoblast and osteoclast activity responsible for bone formation and resorption were evaluated in mouse tail vertebrae using a commercial available molecular fluorescence probe targeting hydroxyapatite. Clear changes in the reconstructed fluorescence intensity were observed for different loading forces and furthermore the FMT readouts was shown to correlate well with a X-ray computed tomography (CT) readout. A further proof demonstrating the feasibility of quantitative FMT imaging was performed on a second experiment where the objective was to image cervical lymph nodes with an indocyanine green (ICG) labeled antibody in an inflamed mouse model. In this study the localization of the inflamed and healthy lymph node was shown with help of reconstructed ICG distribution. Interestingly even signals from the lymph nodes in control animals could be observed at the specific wavelength used. Finally, FMT was applied in a mouse model of Alzheimer’s disease where the time course of fluorescence intensity elicited by an oxazine derived dye, which has been shown to specifically bind to aggregated beta-amyloid in the brain cortex, was compared in transgenic animals developing amyloid plaques to wild type litter mates. The fluorophore concentration could be clearly localized in the cortical region of both animals: the slower dye clearance in the brain of transgenic animals indicated an enhanced dye binding to the Alzheimer’s plaques. In summary, a successful operation of the designed FMT method could be shown by means of various in vivo applications resulting in well resolved reconstructed fluorophore distributions and its quantification under in vivo conditions. A major limitation of FMT, which is characterized by high sensitivity sufficient for providing molecular and functional information, is the relatively poor spatial resolution in comparison to structural/anatomical imaging modalities which may render the allocation of molecular data to a specific anatomic structure difficult. This lead to the second major goal of this project where the elimination of this constraint was intensively studied. The result was a novel hybrid multimodal imaging concept where a combination of non-contact FMT and high field MRI was used for simultaneous measurements. Preclinical MRI is an attractive counterpart to FMT as it provides high resolution images of structures due to excellent soft tissue contrast. This complementary anatomical information would help to better localize and furthermore improve the reconstructed fluorophore distribution. With a non-contact free space FMT design principle restrictions of recently published setups such as low numbers of source/detector pairs in a rigid configuration, fiber based bulky construction, small field of view coverage and related limited diffusive light collection could be eliminated. The here presented newly developed setup is working in non-contact fiber-free reflection geometry where the optical detector array is placed in close proximity to the sample in the isocenter of a 9.4T animal MRI scanner. The sample illumination is provided from outside the bore of the MR scanner and is freely adjustable to the geometry of the sample. First performance tests of the setup in tissue mimicking silicone phantoms using a 32×32 single photon avalanche diode (SPAD) array yielded adequate spatial resolution both in plane and in depth, which correlated well with simulation results. The proof of principle of this first generation setup was demonstrated in a tumor bearing mouse where the distribution of a fluorescent protease probe could be three dimensionally represented and localized with help of an anatomical reference provided by the MR. Based on experiences of the first generation FMT/MRI setup a further improved completely redesigned second generation setup was implemented. The second setup is primed to be used in transmission or reflection mode and the limited detector resolution was extended by using newly developed detector technologies having higher number of pixels available. A specially designed 256×256 CMOS detector array could be attached to the system. The small field of view limiting the optical imaging in the first setup was widened with an improved objective lens and a larger detector size. A further gain in field of view was achieved by increasing the focal distance which was achieved by scanning the sample surface from the side and not as in the previous version from the top. Moreover simplifications for the handling were achieved by attaching the platform to a rigid rail system consisting of two carbon rods fixed to the MR scanner compared to the less stable fixation used in the first version. First tests on tissue mimicking silicone phantoms demonstrated the proper function of both the MR and the optical part during simultaneous acquisitions. Furthermore it was found that the spatial resolution and the sensitivity is comparable to stand alone FMT setups working with the same principle. In vivo experiments in an Alzheimer’s disease mouse model performed simultaneously with the FMT/MRI setup showed comparable results to the presented study with the stand alone FMT system. Summing up, based on a new idea of combining FMT and MRI imaging a first generation and improved second generation dual-modality setup allowing simultaneous measurements was designed and characterized. Performance tests on tissue phantoms and in vivo experiments in mice demonstrated a correct functioning of both modalities and showed the potential of such hybrid setups in providing complementary information required for a comprehensive understanding of the processes investigated.