Development of Optical Tools for the Assessment of Cellular Biomechanics

Wax, Adam P

Eldridge, William J

2020-01-27T16:52:39Z

2020-09-12T08:17:13Z

2019

Biomedical Engineering

Cellular mechanobiology has been of great to scientists, as changes in stiffness has been linked to various biological phenomena. Specifically, variation of cellular viscoelasticity occurs in cancerous cells, likely due to abnormal behavior of the cytoskeleton. Current standard methods for probing cellular stiffness are slow, laborious, and utilize complex and sometimes indirect detection mechanisms that stymy the progress of research in the field. As such, we developed a tool using quantitative phase imaging (QPI) to directly measure mechanical displacement in living cells in response to static loading. In this dissertation, instrumentation and methodologies are developed to probe mechanical differences in living cells, in a typical culture environment, without complex external devices or exogenous contrast agents.

An off-axis quantitative phase microscope was constructed and used to image cells subjected to shear flow within a flow cell. Cellular center-of-mass deviations were fitted to simple one-dimensional, viscoelastic, mechanical models to model cellular deformation and extract a shear stiffness parameter. Cells were tested at multiple flow rates to confirm linear viscoelasticity. Shear stiffness parameters between normal and pharmacologically disrupted cell lines were compared to demonstrate the assay’s ability to segregate different mechanical populations. The assay was then applied to a carcinogenesis model, mimicking transformation of normal bronchial epithelium into carcinogenic phenotypes via intracellular (arsenic) and extracellular (soft agar) changes. Changes in mean stiffness and the relative standard deviation of stiffness were found amongst all groups, indicating mechanical changes occur during oncogenesis.

In addition to the shear flow assay, a method for measuring cellular microstructure via the framework of disorder strength was created using QPI images. Disorder strength was measured by assessing phase variance over small windows across QPI images. Sensitivity to disordered media was verified by phase variance measurements of polystyrene bead solutions. Disorder strength was measured for multiple cell lines, and was found to closely resemble previous measurements. Additionally, disorder strength was found to be strongly correlated to shear stiffness, indicating that inferences of mechanical integrity could be acquired from QPI images alone. To verify these findings and to assert QPI’s utility for mechanical measurements, a QPI method for measuring shear modulus was devised and directly compared to atomic force microscopy (AFM) measurements of Young’s modulus. Shear modulus and disorder strength, via QPI, were measured across six groups of cells and compared to AFM-based Young’s modulus measurements. Comparison of shear modulus and Young’s modulus were found to strongly agree with theory, confirming the integrity of QPI based mechanical measurements. Shear modulus and Young’s modulus were found to be negatively correlated to disorder strength, reinforcing previous findings of a relationship between microstructure and cellular mechanical status. Finally, a combined QPI and fluorescence set up was constructed to add molecular measurements to the morphological imaging capability of QPI. Specifically, FRET based apoptosis sensors were used to monitor morphological parameters of HeLa cells during apoptosis. Cells were found to have optical volume and disorder strength modulation in response to caspase-3 mediated apoptosis. Additionally, cells transfected with FRET-based vinculin tension sensors (VinTS) were analyzed while subjecting cells to static loading. QPI was used to ensure mechanical loading occurred via measured center-of-mass displacements, while VinTS reported the relative tensional changes via changes in FRET index before and after flow subjugation. Indeed, tension was found to increase due to shear flow in response to mechanical displacement, after contributions from focal shifts and photobleaching were accounted for.

These results demonstrated the applicability of QPI as a tool for analyzing cellular mechanical parameters. Additionally, QPI elucidated changes in mechanical status during oncogenic transformation and affirmed the connection between microstructure and mechanical integrity. These measures were confirmed to be valid when compared to gold standard methodologies for cellular mechanical interrogation. Finally, the system was outfitted with molecular imaging capabilities, enabling co-registered measurements of cellular morphological changes with molecular specific changes during apoptosis and static shear loading. In summary, QPI can be an indispensable tool for evaluating mechanical characteristics of cells.

https://hdl.handle.net/10161/19875

Biomedical engineering

Development of Optical Tools for the Assessment of Cellular Biomechanics

Dissertation

7.4958904109589035