Advances in Optical Imaging Techniques for In Vitro 3D Cell Culture and Mechanotransduction Studies

Abstract

Biomedical research is transitioning from traditional two-dimensional (2D) platforms to more complex three-dimensional (3D) cell culture models that more closely mimic <i>in vivo</i> conditions. These models include traditional 3D cultures, organoids, body-on-a-chip, and microphysiological systems. This shift is facilitated by advances in stem cell-derived cell models, novel 3D scaffold biomaterials, and 3D bio-printing and microfluidic chips. However, assessing these <i>in vitro</i> 3D cell culture models requires non-invasive imaging methods capable of providing detailed information at high penetration depths (1-10 mm). Standard optical microscopy techniques, despite their importance in cell and molecular biology labs, fail to provide information at greater depths. Thus, there is a demand for the development of novel, label-free 3D imaging techniques.

The aim of this thesis was to develop novel <i>in vitro</i> techniques for studying 3D cell cultures. These techniques that are capable of providing a non invasive, label- free high-resolution 3D imaging at mesoscopic depths (1 to 10 mm) to bridge the gap between microscopic and macroscopic imaging. This was achieved by developing and optimizing label-free quantitative optical projection tomography (OPT) imaging system. Bright-field OPT, a 3D imaging technique, provides image contrast based on differences in light attenuation within the specimen and achieves isotropic resolution at mesoscopic depths. The method involves acquiring projection images from various angles and applying a mathematical 3D reconstruction algorithm. OPT was designed, constructed and a quantitative imaging protocol was established for measuring cell shape, size, and density within 3D cell cultures. By integrating an electrically tunable lens (ETL) into the OPT setup a multifocal optical projection microscopy (MF-OPM) system was created, which enabled extended depth imaging without the need to translate the specimen or the objective lens. The linear characteristics of the applied current to the ETL and the resulting shift in depth demonstrated the suitability of the integration for multifocal imaging within MF-OPM system. Additionally, both methods provided complementary functional information when used for imaging in fluorescence mode. Finally, using advanced 3D microfabrication via two-photon polymerization and high-resolution imaging to engineer microstructures and study cellular guidance and mechanics.

The functionality of the developed quantitative OPT imaging protocol was validated by analyzing the 3D cellular responses within different hydrogel formulations, focusing on cell morphology, density, distribution, and viability. The analysis revealed distinct differences in the morphological responses of fibroblasts cultured in classical gellan gum (GG) hydrogels compared to those in protein-functionalized gelatin-GG hydrogels. This demonstrates the sensitivity of OPT to detect variations in cellular responses to different material biochemical properties. The developed MF- OPM system was further validated through multifocal imaging of fibroblasts in 3D Geltrex hydrogel. The findings demonstrated the importance of deep imaging, as it provides more detailed information than single focal plane images. Importantly, optical flow analysis showed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) clusters exhibited varying contraction profiles across different focal planes and angles, highlighting the necessity of extended depth and rotation analysis in 3D CM models. Furthermore, substrate microtopographies influenced epithelial cell guidance and alter actin cytoskeleton organization and molecular forces at the nuclear envelope.

In conclusion, this dissertation presents new tools for <i>in vitro</i> 3D imaging at mesoscopic depths. The developed quantitative OPT and MF-OPM imaging systems can be applied to study large volume 3D cell culture models, which are difficult to image with traditional microscopy. Additionally, this work highlights the combined use of TPP and genetically encoded Förster resonance energy transfer (FRET)-based tension biosensors for controlled modulation of topographical features and measurement of tensile tension, respectively. Altogether, these methods offer suitable platforms for the non-destructive, high-resolution quantitative evaluation of biological specimens, thereby advancing our understanding of cellular morphology and mechanics within 3D microenvironments.