Tailoring the Sensitivity of Microcantilevers To Monitor the Mass of Single Adherent Living Cells.

Microcantilevers are widely employed as mass sensors for biological samples, from single molecules to single cells. However, the accurate mass quantification of living adherent cells is impaired by the microcantilever's mass sensitivity and cell migration, both of which can lead to detect masses mismatching by ≫50%. Here, we design photothermally actuated microcantilevers to optimize the accuracy of cell mass measurements. By reducing the inertial mass of the microcantilever using a focused ion beam, we considerably increase its mass sensitivity, which is validated by finite element analysis and experimentally by gelatin microbeads. The improved microcantilevers allow us to instantly monitor at much improved accuracy the mass of both living HeLa cells and mouse fibroblasts adhering to different substrates. Finally, we show that the improved cantilever design favorably restricts cell migration and thus reduces the large measurement errors associated with this effect.

Monitoring the mass, eigenfrequency, and quality factor of mammalian cells.

The regulation of mass is essential for the development and homeostasis of cells and multicellular organisms. However, cell mass is also tightly linked to cell mechanical properties, which depend on the time scales at which they are measured and change drastically at the cellular eigenfrequency. So far, it has not been possible to determine cell mass and eigenfrequency together. Here, we introduce microcantilevers oscillating in the Ångström range to monitor both fundamental physical properties of the cell. If the oscillation frequency is far below the cellular eigenfrequency, all cell compartments follow the cantilever motion, and the cell mass measurements are accurate. Yet, if the oscillating frequency approaches or lies above the cellular eigenfrequency, the mechanical response of the cell changes, and not all cellular components can follow the cantilever motions in phase. This energy loss caused by mechanical damping within the cell is described by the quality factor. We use these observations to examine living cells across externally applied mechanical frequency ranges and to measure their total mass, eigenfrequency, and quality factor. The three parameters open the door to better understand the mechanobiology of the cell and stimulate biotechnological and medical innovations.

An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells.

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced "cell-based molography" as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells.