Developing an Optomechanical Approach for Characterizing Mechanical Properties of Single Adherent Cells

Mechanical properties of a cell reflect its biological and pathological conditions including cellular disorders and fundamental cellular processes such as cell division and differentiation. There have been active research efforts to develop high-throughput platforms to mechanically characterize single cells. Yet, many of these research efforts are focused on suspended cells and use a flow-through configuration. Therefore, adherent cells are detached prior to the characterization, which seriously perturbs the cellular conditions. Also, methods for adherent cells are limited in their throughput. My study is aimed to fill the technical gap in the field of single cell analysis, which is a high-throughput and non-invasive mechanical characterization of single adherent cells. I developed a multi-modal platform to mechanically characterize single adherent cells. The platform is based on optomechanical principle, which induces least perturbation on the cells and does not require cell detachment. Besides, multiple measurements can be performed on a single cell to track its mechanical behavior over time. Proposed platform can expand our understanding on the relationship between mechanical properties and cellular status of adherent cells. Single adherent cells are characterized optomechanically using the vibration-induced phase shift (VIPS). VIPS is a phase shift of apparent velocity of a vertically vibrating substrate measured with laser Doppler vibrometer (LDV), when the measurement laser passes through an adherent cell or any transparent objects on the substrate. The VIPS and height oscillation of a single cell on a vibrating substrate have negative correlation with the cell stiffness. An analytical model is established which demonstrates relationship between cell’s mechanical properties and its VIPS. With the VIPS measurements, at multiple frequencies on large population of cells, the statistical significant difference in the cell stiffness is confirmed after exposure to various drugs affecting cytoskeleton network. Also, a 3-dimensional finite element model is developed to extract the cell stiffness from VIPS. VIPS technique is used to reconstruct the detailed oscillation pattern of transparent objects such as water microdroplets and intracellular lipid droplets on a vibrating substrate, which can give us better understanding of mechanical behavior of biological transparent objects. In addition, using VIPS measurement mechanical interaction between extracellular matrixes (ECMs) and adherent cells is studied. Statistical significant difference in bonding straight of single cells and different ECMs is demonstrated

Cell viscoelasticity is linked to fluctuations in cell biomass distributions.

The viscoelastic properties of mammalian cells can vary with biological state, such as during the epithelial-to-mesenchymal (EMT) transition in cancer, and therefore may serve as a useful physical biomarker. To characterize stiffness, conventional techniques use cell contact or invasive probes and as a result are low throughput, labor intensive, and limited by probe placement. Here, we show that measurements of biomass fluctuations in cells using quantitative phase imaging (QPI) provides a probe-free, contact-free method for quantifying changes in cell viscoelasticity. In particular, QPI measurements reveal a characteristic underdamped response of changes in cell biomass distributions versus time. The effective stiffness and viscosity values extracted from these oscillations in cell biomass distributions correlate with effective cell stiffness and viscosity measured by atomic force microscopy (AFM). This result is consistent for multiple cell lines with varying degrees of cytoskeleton disruption and during the EMT. Overall, our study demonstrates that QPI can reproducibly quantify cell viscoelasticity

Mechanical oscillations enhance gene delivery into suspended cells

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Simultaneous real-time viscoelasticity, mass and cell cycle monitoring for single adherent cancer cells

Cancer is a complex disease caused by the combined effects of genetic and environmental factors. Evidently, there exists a correlation between the surrounding environment of a cell, its biophysical properties and health. Information gained from biomechanics has led to an improved understanding of the way diseases evolve and their progression cycle, providing methods targeted towards curing these diseases. Countless studies have been carried out on the mechanisms underlying cell cycle progression. More particularly, these studies on the mechanics of individual cells have pointed to their coordination, which helps us understand cellular metabolic and physiological process better. Development of more precise, versatile and reliable measurement tools and techniques will provide a greater understanding of cellular behavior and biophysical properties. Micromechanical systems (MEMS) technology can provide these tools – for analyzing single cells and providing important and useful information of their biophysical properties. In modern research, the ability to reliably investigate and understand these cellular properties requires measurement devices that provide high sensitivity, high throughput, and adaptability to include multiple on-chip functionalities. Many MEMS-based resonant sensors have been extensively studied and used as biological and chemical sensors. However, previous works have shown that there are several technology limitations that inhibit application of various MEMS-sensors to mechanical measurement and analysis, including insufficient cell capture efficiency, media perfusion for long term growth, cell adhesion and cell movement/spreading and cell-sensor modelling. Cellular mechanics and viscoelastic properties are known to play a role in biological processes such as cell growth, stem cell differentiation, cell crawling, wound healing, protein regulation, cell malignancy and even apoptosis (programmed cell death). Thus, an accurate measurement of stiffness and growth is fundamental to understanding cellular proliferation in cancer. Capturing these biophysical properties of cancer cells over the duration of their growth cycle through MEMS devices can help provide a better insight into the mechanics of the metastasis of cancer cells. Meanwhile, many MEMS sensing devices still require further development and characterization to reliably investigate long-term cell behaviors. This dissertation focuses on characterization of our MEMS resonant sensors to address current challenges in the measurement of long-term biophysical behaviors of cells across its cell cycle. The amplitude and frequency of MEMS resonant pedestal sensors were used in conjunction with a vibration induced and optically-sensed phase shift of target light incident on an adhering sample to extract the loss tangent - a measure of the relative viscoelasticity of soft materials. This observed phase shift, combined with a representative two-degree-of-freedom Kelvin-Voigt model, is used to simultaneously obtain the elasticity (stiffness), viscosity and mass associated with individual adherent cancer cells. The research is unique as it decouples the heterogeneity of individual cells in our population and further refines our viscoelastic solution space. This novel development enables long-term simultaneous measurement of changes in stiffness and mass of normal and cancerous cells over time. This is the first investigation of the time-varying simultaneous measurement of viscoelasticity and mass for individual adherent cells using our MEMS resonant sensors

Force and Compliance Measurements on Living Cells Using Atomic Force Microscopy (AFM)

We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study

Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

This work was supported in part by a Marie Curie CIG grant (PCIG14-GA-2013-631011 CSKFingerprints)