Visualization and manipulation of repair and regeneration in biological systems using light

Abstract

Tissue repair after an injury is a fundamental process in biomedicine. It can involve regeneration, which uses new growth to restore tissue function. The interest in repair and regeneration is motivated by the desire to treat injuries and diseases and has attracted researchers for centuries. In the last decades, it evolved in the field of regenerative medicine, which has the ultimate goal of providing strategies for regenerating human cells, tissues, or even organs, for instance, via engineering principles.

Already since the first experiments on regeneration by Abraham Trembley, novel findings in biomedicine, repair, and regeneration have been enabled or accompanied by research in optics, for example, on the development of novel microscopy techniques. Nowadays, novel optical techniques are advancing, which allow to understand the role of single cells in tissue repair processes. Moreover, repair processes within cells can be visualized and manipulated. Ultimately, optics can provide enabling techniques for regenerative therapies. This habilitation thesis aims to present several of these advances.

On a single cell level, femtosecond laser nanosurgery was used to target specific intracellular structures during concurrent imaging in vitro. The relation of femtosecond laser nanosurgery to the cell state and cellular staining was investigated. Manipulation of single Z-discs in cardiomyocytes using a femtosecond oscillator laser system was accomplished, which allows to better elucidate the role of a single Z-disc in cardiomyocyte function. In particular, measurements on cell survival, (calcium-) homeostasis, and morphology yielded only minor deviations from control cells after single Z-disc ablation. A reduction in force generation was elucidated via traction force microscopy and gene expression level changes, for instance, an upregulation of -actinin were examined.

Additionally, light-based systems to influence single cells in their alignment or to trigger single cells, for example, to activate other cells via optogenetics were applied. On the tissue scale, imaging via confocal microscopy or multiphoton microscopy has been applied for various contexts of regenerative approaches. Furthermore, a fiber-based imaging approach, which could later be used for longitudinal imaging in vivo and builds upon a fluorescence microscope system and an imaging fiber bundle in combination with reconstruction via a neural network, was developed. As another imaging strategy, an abdominal imaging window served to image the mouse liver in vivo via multiphoton microscopy in successive imaging sessions. Manipulation in tissue was applied in colonoids, which resemble the structure of the colon on an in vitro scale, and revealed different cell dynamics dependent on the location of the damage. In particular, activation of the Wnt signaling pathway after crypt damage was observed. Cell ablation via a femtosecond laser amplifier system during concurrent two-photon microscopy was also established during in vivo liver imaging to study micro-regenerative processes.

Furthermore, laser-based delivery processes with novel materials or in the context of genome editing using CRISPR/Cas9 technology were investigated as enabling technologies for regenerative medicine. In conclusion, this thesis addresses the question of how optics can help to illuminate future directions in research on tissue repair and regeneration, as well as, regenerative therapies by addressing (longitudinal) imaging in a complex environment, sophisticated cell-manipulation strategies, and the application of novel materials for laser-based delivery.