The Nanoscale World

Nanomechanical measurements have been used increasingly to evaluate the cellular effects of diseases and various drugs, and even to differentiate between cancerous and non-cancerous cells. Without question, the nanoscale analysis of cell mechanics provides an exciting emerging method for analyzing a variety of biological and biochemical responses in cells. Jason Reed and his colleagues at the California Nanosystems Institute (CNSI) at UCLA have been using an interference microscope to study cellular nanomechanics, and have developed a customized system that exhibits unique benefits in understanding and predicting pathology at a cellular level.

There are currently a number of traditional techniques for quantifying cell biophysical properties, including atomic force microscopy (AFM), bio-microrheology (BMR), magnetic twisting rheometry, microplate stretching, optical tweezers, optical stretching, and micropipette aspiration. These approaches have provided new insights into the structure and function of the intracellular matrix, cytoskeleton, and cell membrane, and also have shown the potential for characterizing and classifying diseased cell states. Study results suggest the potential existence of a detectable, quantifiable mechanical signature for cell types and diseased states.^1-3

However, certain limitations of the common nanomechanical measurement techniques above must be overcome, such as the inability to measure extremely soft materials, speed, rheological changes from the methods, destructivity, and weak linking between the measured parameter and cell mechanics. To resolve some of these issues, Reed’s group worked with Veeco engineers to develop a new, relatively high-throughput cell analysis system. The system employs Veeco’s Wyko-brand interference microscope for non-destructive, near real-time measurements of cell nanomechanical responses to force application or drugs, without the need for staining or other labeling techniques. For the observation of cells in a fluid by the optical profiler, a special reference cell was constructed for Veeco’s through-transmissive media objective that matched exactly the dispersion of the liquid cell media. Finally, a live cell perfusion chamber was built with a fixed viewing window for observation.

With this technology, mechanical properties of cells can be assessed in situ and in parallel using micro-indentation techniques. Samples are prepared for imaging by placing microreflectors on top of cells as probes for measuring nanometer displacements in cell height and position. The mechanical imaging interferometry provides direct biophysical data, such as cell thickness and position, for approximately 1000 cells at any given time. Nanomechanical properties, such as the Young’s Modulus of cells, can be ascertained by imparting a magnetic field on the microreflectors (20pN – 20nN) to measure force displacement on the surface of the cells.

Reed’s initial studies evaluated the efficacy of using interferometry in live cell studies.^3-5 The figure below of human hct116 colon carcinoma cells in a culture medium shows considerable detail of the cell membrane, including intra-cellular organelles. This measurement represents the variations in the optical path as the incident beam travels through the fluid and the almost completely transparent cell. This signature is linear with the optical thickness of the cell because the whole wavefront traveling through the cell is interfered with the independent reference wavefront, and thus this type of measurement is called quantitative phase imaging. Using this method, Reed et al. found that the nanomechanics in hundreds of cells can be monitored simultaneously at a vertical resolution of <50 nanometers. In addition, they were able to achieve high-throughput measurement of changes in the viscoelastic behavior of treated cells.

In another recent study, local redistribution of cell content was monitored as small indentions were made by highly magnetic probes on the cell surface. It was found that there was almost instantaneous redistribution of cell material as a result of indentation on the surface of the cell, which was undetected with intensity imaging alone. Secondly, changes in local compliance were observed within 200 seconds when force was applied cyclically to regions of the cell. It would be extremely difficult to measure these types of immediate viscoelastic measurements with conventional biophysical measurement techniques, particularly at such a large scale.

In order to understand and predict pathways toward effective disease therapy, there is a need for novel non-invasive means for establishing molecular signatures for disease and evaluating cell response to treatments. Reed’s new interferometric approach enables high-throughput, large-scale measurement of cellular nanomechanical responses, with the potential to identify and monitor specific cells within a bulk population. These features are desirable for any system that is used to distinguish between two cell types within a heterogeneous cell mixture, or even to identify cancer stem cells.

References

1. Guck, J., Schinkinger, S., Lincoln, B., Wottawah, F., Ebert, S., Romeyke, M. et al. (2005). Optical Deformability as an Inherent Cell Marker for Testing Malignant Transformation and Metastatic Competence. Biophys. J 88(5), 3689-98.

2. Elson, E., L. (1988). Cellular Mechanics as an Indicator of Cytoskeletal Structure and Function. Annual Review of Biophysics and Biophysical Chemistry 17(1), 397-430.

3. Reed, J., Walczak, W. J., Petzold, O. N., Gimzewski, J. K. (2009). In Situ Mechanical Interferometry of Matrigel Films. Langmuir 25(1), 36-39.

4. Reed, J., Troke, J. J., Schmit, J., Han, S., Teitell, M. A., Gimzewski, J. K. (2008). Live Cell Interferometry Reveals Cellular Dynamism During Force Propagation.ACS Nano 2(5), 841-46.

5. Reed, J., Frank, M., Troke, J., J., Schmit, J., Han, S., Teitell, M., A. et al. (2008). High Throughput Cell Nanomechanics with Mechanical Imaging Interferometry.Nanotechnology 19(23), 235101.