Multispectral methods for biological sensing and imaging. Theory and experiments.  

Multispectral molecular imaging exhibits the unique potential to simultaneously probe multiple molecules in living objects. This thesis reports on developments realized on methodological and systems level that aim at applying multispectral concepts to molecular imaging. Research was directed towards various application areas, from surface investigations of intrinsic tissue molecules to volumetric imaging of extrinsic molecular markers. The presented results show the capability of multispectral imaging to asses multiple molecules in-vivo, a property which is of tremendous value in systemic biomedical research.&nbsp

Quantitative multi-spectral oxygen saturation measurements independent of tissue optical properties.

Imaging of tissue oxygenation is important in several applications associated with patient care. Optical sensing is commonly applied for assessing oxygen saturation but is often restricted to local measurements or else it requires spectral and spatial information at the expense of time. Many methods proposed so far require assumptions on the properties of measured tissue. In this study we investigated a computational method that uses only multispectral information and quantitatively computes tissue oxygen saturation independently of tissue optical properties. The method is based on linear transformations of measurements in three isosbestic points. We investigated the ideal isosbestic point combination out of six isosbestic points available for measurement in the visible and near-infrared region that enable accurate oxygen saturation computation. We demonstrate this method on controlled tissue mimicking phantoms having different optical properties and validated the measurements using a gas analyzer. A mean error of 2.9 ± 2.8% O2 Sat was achieved. Finally, we performed pilot studies in tissues in-vivo by measuring dynamic changes in fingers subjected to vascular occlusion, the vasculature of mouse ears and exposed mouse organs. Selected steps of spectral transformations applied to oxygenation spectra. The original reflectance spectrum M(λ) is transformed in step 1 to overlap with reference spectra (grey) in three isosbestic points, resulting in M″(λ). In step 2, the gradient of M″(λ) is computed resulting in M″grad (λ), which can be used for quantitative oxygenation computation

Improving limited-projection-angle fluorescence molecular tomography using a co-registered X-ray computed tomography scan.

We examine the improvement in imaging performance, such as axial resolution and signal localization, when employing limited-projection-angle fluorescence molecular tomography (FMT) together with x-ray computed tomography (XCT) measurements versus stand-alone FMT. For this purpose, we employed living mice, bearing a spontaneous lung tumor model, and imaged them with FMT and XCT under identical geometrical conditions using fluorescent probes for cancer targeting. The XCT data was employed, herein, as structural prior information to guide the FMT reconstruction. Gold standard images were provided by fluorescence images of mouse cryoslices, providing the ground truth in fluorescence bio-distribution. Upon comparison of FMT images versus images reconstructed using hybrid FMT and XCT data, we demonstrate marked improvements in image accuracy. This work relates to currently disseminated FMT systems, using limited projection scans, and can be employed to enhance their performance

Limited-projection-angle hybrid fluorescence molecular tomography of multiple molecules.

ABSTRACT. An advantage of fluorescence methods over other imaging modalities is the ability to concurrently resolve multiple moieties using fluorochromes emitting at different spectral regions. Simultaneous imaging of spectrally separated agents is helpful in interrogating multiple functions or establishing internal controls for accurate measurements. Herein, we investigated multimoiety imaging in the context of a limited-projection-angle hybrid fluorescence molecular tomography (FMT), and x-ray computed tomography implementation and the further registration with positron emission tomography (PET) data. Multichannel FMT systems may image fluorescent probes of varying distribution patterns. Therefore, it is possible that different channels may require different use of priors and regularization parameters. We examined the performance of automatically estimating regularization factors implementing priors, using data-driven regularization specific for limited-projection-angle schemes. We were particularly interested in identifying the implementation variations between hybrid-FMT channels due to probe distribution variation. For this reason, initial validation of the data-driven algorithm on a phantom was followed by imaging different agent distributions in animals, assuming superficial and deep seated activity. We further demonstrate the benefits of combining hybrid FMT with PET to gain multiple readings on the molecular composition of disease

Uniqueness in multispectral constant-wave epi-illumination imaging.

Multispectral tissue imaging based on optical cameras and continuous-wave tissue illumination is commonly used in medicine and biology. Surprisingly, there is a characteristic absence of a critical look at the quantities that can be uniquely characterized from optically diffuse matter by multispectral imaging. Here, we investigate the fundamental question of uniqueness in epi-illumination measurements from turbid media obtained at multiple wavelengths. By utilizing an analytical model, tissue-mimicking phantoms, and an in vivo imaging experiment we show that independent of the bands employed, spectral measurements cannot uniquely retrieve absorption and scattering coefficients. We also establish that it is, nevertheless, possible to uniquely quantify oxygen saturation and the Mie scattering power-a previously undocumented uniqueness condition

Integration of metabolic databases for the reconstruction of genome-scale metabolic networks.

Background: Genome-scale metabolic reconstructions have been recognised as a valuable tool for a variety of applications ranging from metabolic engineering to evolutionary studies. However, the reconstruction of such networks remains an arduous process requiring a high level of human intervention. This process is further complicated by occurrences of missing or conflicting information and the absence of common annotation standards between different data sources. Results: In this article, we report a semi-automated methodology aimed at streamlining the process of metabolic network reconstruction by enabling the integration of different genome-wide databases of metabolic reactions. We present results obtained by applying this methodology to the metabolic network of the plant Arabidopsis thaliana. A systematic comparison of compounds and reactions between two genome-wide databases allowed us to obtain a high-quality core consensus reconstruction, which was validated for stoichiometric consistency. A lower level of consensus led to a larger reconstruction, which has a lower quality standard but provides a baseline for further manual curation. Conclusion: This semi-automated methodology may be applied to other organisms and help to streamline the process of genome-scale network reconstruction in order to accelerate the transfer of such models to applications

Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: A performance overview.

PURPOSE: A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. PROCEDURES: Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). RESULTS: It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. CONCLUSIONS: We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents

Cardioprotective C-kit⁺ bone marrow cells attenuate apoptosis after acute myocardial infarction in mice &ndash; <em>in-vivo</em> assessment with fluorescence molecular imaging.

Cardiomyocyte loss via apoptosis plays a crucial role in ventricular remodeling following myocardial infarction (MI). Cell-based therapy approaches using bone marrow derived c-kit(+) pluripotent cells may attenuate apoptosis following ischemic injury. We therefore thought to examine the early course of apoptosis following myocardial infarction - in-vivo - and non-invasively determine the effect of c-kit(+) bone marrow cells on post-MI remodeling. We studied apoptosis in wild-type Kit(+/+), c-kit mutant Kit(W)/Kit(W-v) and Kit(W)/Kit(W-v) mice after cell therapy with bone-marrow derived c-kit(+) cells after ischemia-reperfusion injury. Mice were followed by hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) at 6h, 24h and 7 days after ischemia-reperfusion injury using an Annexin V-based fluorescent nanosensor targeting phosphatidylserine. Kit(W)/Kit(W-v) mice showed increased and prolonged apoptosis compared to control Kit(+/+) mice while c-kit cell therapy was able to attenuate the altered apoptosis rates. Increased apoptosis was accompanied by severe decline in heart function, determined by cardiac Magnetic Resonance Imaging, and cell therapy was able to rescue the animals from deleterious heart failure. Post-mortem cryoslicing and immunohistochemistry localized the fluorescence signal of the Annexin V sensor within the infarcted myocardium. Flow cytometry of digested infarct specimens identified apoptotic cardiomyocytes as the major source for the in-vivo Annexin V signal. In-vivo molecular imaging using hybrid FMT-XCT reveals increased cardiomyocyte apoptosis in Kit(W)/Kit(W-v) mice and shows that c-kit(+) cardioprotective cells are able to attenuate post-MI apoptosis and rescue mice from progressive heart failure

FMT-PCCT: Hybrid fluorescence molecular tomography-X-ray phase-contrast CT imaging of mouse models.

The implementation of hybrid fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high fidelity and quantification accuracy. FMT affords highly sensitive three-dimensional imaging of fluorescence bio-distribution throughout animal bodies but in stand-alone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from X-ray computed tomography (CT). Conversely, X-ray CT generally suffers from low soft tissue contrast. Therefore utilization of X-ray CT data as prior information to the fluorescence inversion problem is challenging in applications where different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast computed tomography (PCCT). PCCT relies on the phase shift differences in tissue to deliver anatomical images of biological samples with superior soft tissue contrast than conventional absorption-based CT. We demonstrate for the first time hybrid FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively. The results show that FMT-PCCT expands the potential and utility of FMT in imaging lesions which show otherwise low or no contrast in conventional CT, while retaining the cost benefits and technical simplicity of CT and single hybrid devices. The results point to the most accurate hybrid FMT performance to date