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[Image: see text] There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, (99m)Tc-UBI(29–41)-Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with (99m)Tc-UBI(29–41)-Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the (99m)Tc-UBI(29–41)-Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with (99m)Tc-UBI(29–41)-Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.

Cell-based therapies are currently being developed for applications in both regenerative medicine and in oncology. Preclinical, translational, and clinical research on cell-based therapies will benefit tremendously from novel imaging approaches that enable the effective monitoring of the delivery, survival, migration, biodistribution, and integration of transplanted cells. Magnetic resonance imaging (MRI) offers several advantages over other imaging modalities for elucidating the fate of transplanted cells both preclinically and clinically. These advantages include the ability to image transplanted cells longitudinally at high spatial resolution without exposure to ionizing radiation, and the possibility to co-register anatomical structures with molecular processes and functional changes. However, since cellular MRI is still in its infancy, it currently faces a number of challenges, which provide avenues for future research and development. In this review, we describe the basic principle of cell-tracking with MRI; explain the different approaches currently used to monitor cell-based therapies; describe currently available MRI contrast generation mechanisms and strategies for monitoring transplanted cells; discuss some of the challenges in tracking transplanted cells; and suggest future research directions.

Recently, second harmonic generation (SHG) nanomaterials have been generated that are efficiently employed in the classical (NIR) and extended (NIR-II) near infrared windows using a multiphoton microscope. The aim was to test bismuth ferrite harmonic nanoparticles (BFO-HNPs) for their ability to monitor pulmonary macrophages in mice. BFO-loaded MH-S macro phages are given intratracheally to healthy mice or BFO-HNPs are intranasally instilled in mice with allergic airway inflammation and lung sections of up to 100 μM are prepared. Using a two-photon-laser scanning microscope, it is shown that bright BFO-HNPs signals are detected from superficially localized cells as well as from deep within the lung tissue. BFO-HNPs are identified with an excellent signal-to-noise ratio and virtually no background signal. The SHG from the nanocrystals can be distinguished from the endogenous collagen–derived SHG around the blood vessels and bronchial structures. BFO-HNPs are primarily taken up by M2 alveolar macro phages in vivo. This SHG imaging approach provides novel information about the inter action of macrophages with cells and the extracellular matrix in lung disease as it is capable of visualizing and tracking NP-loaded cells at high resolution in thick tissues with minimal background fluorescence.

Medical imaging has the potential to significantly improve our capabilities to understand, diagnose and treat diseases. Novel strategies include cell- tracking with non-targeted contrast agents in basic research and molecular imaging with targeted contrast agents for clinical applications. Both strategies rely on the sensitive detection of labeled cells by non-invasive modalities such as PET, SPECT, CT and MRI. Among these, MRI is the only modality that avoids the use of ionizing radiation. Nevertheless, it suffers from the low sensitivity of conventional relaxivity-based contrast agents (ca. 10-4 M detection limit). Molecular imaging applications that require nanomolar sensitivity are therefore restricted. Hyperpolarized xenon MRI in combination with indirect detection of xenon nanocarriers (hyper-CEST) overcomes this limitation by a 10⁷-fold signal gain. The MRI signal of the noble gas xenon is therefore dramatically enhanced by laser induced spin exchange optical pumping (SEOP) which further allows for the in vivo localization of dissolved xenon within organs such as the brain and the heart following inhalation. In addition, different synthetic as well as biological structures that serve as temporal hosts for xenon atoms have been identified. These nanocarriers are ideal contrast agents for xenon MRI since their indirect detection by chemical exchange saturation transfer combines high sensitivity (nano to picomolar concentrations) with the potential for multiplexing, functionalization and even genetic encoding. Within this study, substantial progress for cell- labeling with synthetic xenon nanocarriers including cryptophane-A cages (CrA) and perfluorcarbon nanodroplets (PFOB) could be achieved. Unspecific cell- labeling with unmodified CrA and PFOB was established as a labeling scheme for potential cell-tracking applications. Specific cell-labeling for the purpose of molecular imaging was confirmed for three different functionalized CrA molecules targeting cell surface epitopes (CD-14, EGF-receptors and metabolically labeled glycans). The functionalization strategies include modular antibody conjugation, scaffolding as well as the coupling of a bioorthogonal group. It was demonstrated for the first time, that the achievable labeling concentrations (micromolar to nanomolar) are indeed sufficient for the in vitro MRI localization of labeled cells by exploiting the signal amplification of indirect hyper-CEST detection. Further on, the frequency selectivity of the hyper-CEST principle has been employed for the multiplexed detection of CrA- and PFOB labeled cells. The multiplexing concept was demonstrated for non-targeted xenon nanocarriers and can be translated to specific labeling applications with functionalized versions in the future. Final in vitro MRI experiments have been performed in an advanced imaging setup (bioreactor) under physiological conditions with live cells. The setup simulates in vivo-like xenon delivery by cell perfusion with xenon saturated medium. All achievements were further discussed with respect to recent developments within the field. The thesis therefore provides a detailed in vitro characterization of MRI cell-labeling with xenon nanocarriers and thereby addresses the fundamental need for a successful translation of the concept to preclinical cell-tracking and molecular imaging applications., Neue Konzepte auf dem Gebiet der medizinischen Bildgebung haben das Potential unsere Möglichkeiten zum Verständnis, der Diagnose und der Heilung von Krankheiten entscheidend zu verbessern. Diese Strategien beinhalten sowohl die Zellverfolgung mittels nicht-zielgerichteter Kontrastmittel im Bereich der Grundlagenforschung, als auch molekulare Bildgebung mit Hilfe von zielgerichteten Kontrastmitteln für klinische Anwendungen. Beide Verfahren basieren auf der sensitiven Detektion von markierten Zellen mittels nicht- invasiver Bildgebungsmethoden wie PET, SPECT, CT und MRT. Von diesen ist MRT das einzige Verfahren, welches nicht auf dem Einsatz ionisierender Strahlung beruht. Die Verwendung von MRT im Bereich der molekularen Bildgebung ist jedoch aufgrund der relativ geringen Sensitivität konventioneller, relaxations-basierter Kontrastmittel limitiert. MRT von hyperpolarisiertem Xenon in Kombination mit der indirekten Detektion von Xenon Nanoträgern (hyper-CEST) ermöglicht es, diese Beschränkung aufgrund einer etwa 107-fachen Signalverstärkung zu überwinden. Das MRT-Signal des ungiftigen Edelgases Xenon wird zu diesem Zweck erheblich mittels laser-induziertem “spin exchange optical pumping (SEOP)“ verstärkt. Diese Signalverstärkung ermöglicht es anschließend, die Verteilung von gelöstem Xenon in Organen wie dem Gehirn oder dem Herzen, nach dessen Inhalation zu detektieren. Zudem wurden verschiedene synthetische als auch biologische Strukturen identifiziert, die als temporärer Wirt für Xenonatome fungieren. Diese Nanoträger stellen ideale Kontrastmittel für Xenon-MRT dar, da ihre indirekte Lokalisierung mittels “chemical exchange saturation transfer“ (CEST) hohe Sensitivität (nano- bis pikomolar) mit der Möglichkeit zur Mehrfachdetektion, Funktionalisierung und genetischen Kodierung verbindet. Im Rahmen dieser Arbeit konnten erhebliche Fortschritte bei der Markierung von Zellen mit synthetischen Xenon-Nanoträgern erzielt werden. Bei den verwendeten Nanoträgern handelt es sich um Cryptophan-A Käfige (CrA) und Perfluorcarbon-Nanotröpfchen (PFOB). Unspezifische Zellmarkierung mit nicht-modifiziertem CrA und PFOB wurde als eine Markierungsstrategie für potentielle Anwendungen der Zellverfolgung etabliert. Spezifische Markierung von zellulären Oberflächenstrukturen (CD-14, EGF-Rezeptor, metabolisch markierte Glykane) zum Zwecke der molekularen Bildgebung wurde für drei unterschiedlich funktionalisierte CrA-Konjugate nachgewiesen. Die verwendeten Funktionalisierungsstrategien umfassen die modulare Konjugation mit Antikörpern, den Einsatz von zielgerichteten Gerüststrukturen sowie die Kopplung einer bioorthogonalen Gruppe. Es wurde zum ersten Mal gezeigt, dass die erzielbaren Markierungskonzentrationen (mikromolar bis nanomolar) in der Tat ausreichend sind, um die in vitro MRT Lokalisierung von markierten Zellen unter Ausnutzung der Signalverstärkung durch indirekte hyper-CEST Detektion zu ermöglichen. Des Weiteren wurde die Frequenzselektivität des hyper-CEST Prinzips zur Mehrfachdetektion von CrA- und PFOB markierten Zellen eingesetzt. Das Konzept wurde für nicht-modifizierte Xenon-Nanoträger demonstriert und kann in der Zukunft auf Anwendungen mit funktionalisierten Nanoträgern übertragen werden. Weiterführende in vitro MRT-Experimente wurden in einem neu entwickelten Bildgebungsaufbau (Bioreaktor) mit lebenden Zellen und unter physiologischen Bedingungen durchgeführt. Der Aufbau simuliert die in vivo- Anlieferung von Xenon durch Zellperfusion mit Xenon-gesättigtem Medium. Die vorgelegte Studie liefert die Grundlagen für eine detaillierte in vitro- Charakterisierung der MRT-Zellmarkierung mit Xenon-Nanoträgern. Eine solche Charakterisierung ist die Vorrausetzung für eine erfolgreiche Übertragung des Konzeptes hin zu präklinischen Anwendungen auf dem Gebiet der Zellverfolgung und der molekularen Bildgebung.

In the present study complementary high-resolution imaging techniques on different length scales are applied to elucidate a cellular loading protocol of gold nanoparticles and subsequently its impact on long term and high-resolution cell-tracking utilizing X-ray technology. Although demonstrated for malignant cell lines the results can be applied to non-malignant cell lines as well. In particular the accumulation of the gold marker per cell has been assessed quantitatively by virtue of electron microscopy, two-dimensional X-ray fluorescence imaging techniques and X-ray CT with micrometric and sub-micrometric resolution. Moreover, utilizing these techniques the three dimensional distribution of the incorporated nanoparticles, which are sequestered in lysosomes as a permanent marker, could be determined. The latter allowed elucidation of the gold partition during mitosis and the cell size, which subsequently enabled us to define the optimal instrument settings of a compact microCT system to visualize gold loaded cells. The results obtained demonstrate the feasibility of cell-tracking using X-ray CT with compact sources.

The neural crest is a transient population of cells that migrate away from the dorsal neural tube in the vertebrate embryo. As the developing hindbrain constricts into rhombomeres, cranial neural crest cells migrate in three discrete streams adjacent to even-numbered rhombomeres, rhombomere 2 (r2), r4, and r6. To test the role of intrinsic versus extrinsic cues in influencing an individual cell’s trajectory, we implanted physical barriers in the chick mesoderm, distal to emerging neural crest cells (NCCs). We analyzed spatio-temporal dynamics as NCCs encountered and responded to the barriers by using time-lapse confocal microscopy and cell tracking analysis. The majority of NCCs were able to overcome physical barriers. Even though the lead cells become temporarily blocked by a barrier, follower cells find a novel pathway around a barrier and become de novo leaders of a new stream. Quantitative analyses of cell trajectories find cells that encounter an r3 barrier migrate significantly faster but less directly than cells that encounter an r4 barrier, which migrate normally. NCCs can also migrate into normally repulsive territory as they reroute. These results suggest that cranial neural crest cell trajectories are not intrinsically determined. NCCs can respond to minor alterations in the environment to retarget a peripheral destination. Both intrinsic and extrinsic cues are important in patterning. We then tested the role of Eph/ephrin signaling on cranial neural crest migration by ectopically expressing full-length ephrin-A5 ligand; a truncated, constitutively active EphA4 receptor; and a truncated, kinase-dead EphA4 receptor within migratory neural crest cells. Ectopic expression of ephrin-A5 specifically causes the r6 subpopulation of neural crest cells to have truncated migration but does not affect directionality, suggesting that the r6 neural crest cells properly follow guidance cues. Our results support a role for ephrin-A5 in regulating the extent of migration. Ectopic expression of constitutively active, truncated EphA4 causes NCCs to migrate aberrantly around the otic vesicle. Pathfinding errors are accompanied by changes in migratory behavior, with the NCCs migrating faster but with less directionality. Expression of a truncated, kinase-dead version of EphA4 also leads to pathfinding errors. Our results suggest Eph activity is involved in guidance and extent of migration.

Somitogenesis is the rhythmic and sequential formation of somites, which are tissue blocks that give rise to segmented adult body structures including the vertebrae and associated muscle. Somite formation is controlled by the segmentation clock, a population of genetic oscillators that are coordinated by an interplay of cell-intrinsic and -extrinsic spatiotemporal information. Disruption of the segmentation clock can lead to misplaced or defective somite boundaries, and consequently results in deformed adult structures (e.g., congenital scoliosis). Despite decades of research into how the segmentation clock pattern is established, and how it acts to position somite boundaries within the pre-segmental mesoderm(PSM), many open questions remain. The position where the somite boundary is set along the anteroposterior axis of the PSM has been named the "determination front". Question still remain as to the mechanism and location of the determination front, and what spatiotemporal information is instructive. Here I present three studies that tackle this question by advancing imaging and analysis tools such that questions that have persisted for decades can be directly addressed. My work contributed significantly to obtaining a better picture of how somite boundaries are precisely formed.

The fundamental basis in the development of novel radiotherapy methods is in-vitro cellular studies. To assess different endpoints of cellular reactions to irradiation like proliferation, cell cycle arrest, and cell death, several assays are used in radiobiological research as standard methods. For example, colony forming assay investigates cell survival and Caspase3/7-Sytox assay cell death. The major limitation of these assays is the analysis at a fixed timepoint after irradiation. Thus, not much is known about the reactions before or after the assay is performed. Additionally, these assays need special treatments, which influence cell behavior and health. In this study, a completely new method is proposed to tackle these challenges: A deep-learning algorithm called CeCILE (Cell Classification and In-vitroLifecycle Evaluation), which is used to detect and analyze cells on videos obtained from phase-contrast microscopy. With this method, we can observe and analyze the behavior and the health conditions of single cells over several days after treatment, up to a sample size of 100 cells per image frame. To train CeCILE, we built a dataset by labeling cells on microscopic images and assign class labels to each cell, which define the cell states in the cell cycle. After successful training of CeCILE, we irradiated CHO-K1 cells with 4 Gy protons, imaged them for 2 days by a microscope equipped with a live-cell-imaging set-up, and analyzed the videos by CeCILE and by hand. From analysis, we gained information about cell numbers, cell divisions, and cell deaths over time. We could show that similar results were achieved in the first proof of principle compared with colony forming and Caspase3/7-Sytox assays in this experiment. Therefore, CeCILE has the potential to assess the same endpoints as state-of-the-art assays but gives extra information about the evolution of cell numbers, cell state, and cell cycle. Additionally, CeCILE will be extended to track individual cells and their descendants throughout the whole video to follow the behavior of each cell and the progeny after irradiation. This tracking method is capable to put radiobiologic research to the next level to obtain a better understanding of the cellular reactions to radiation.