Applications of scanning ion conductance microscopy (sicm) in cytomechanics
A current challenge in the life sciences is to understand how biological systems change their structural, biophysical and chemical properties to adjust functionality. Cellular responses to mechanical forces are crucial in embryonic development and adult physiology, and are involved in numerous diseases, including atherosclerosis, hypertension, osteoporosis, muscular dystrophy, myopathies and cancer.
Atomic Force Microscopy (AFM) is commonly used to investigate local mechanical properties of cells. However, AFM imaging resolution on cells is generally limited to about 50 nanometers because of the soft, fragile, corrugated and dynamic nature of the cell surface, which is partially deformed by the AFM tip. In addition, the tip is subject to contamination by loosely bound macromolecules (glycocalyx).
Scanning Ion Conductance Microscopy (SICM) is a scanning probe technique, working in ionic solution, in which the probe is not in direct contact with the surface. This can be achieved by scanning a nanopipette over the specimen surface while monitoring the ionic current flowing between the electrode inside the pipette and a reference electrode in the bath. In our work “Measuring the elastic properties of living cells through the analysis of current–displacement curves in scanning ion conductance microscopy”, pulished in Pflugers Archive – European Journal of Physiology in 2012, we have shown that the SICM technique is also capable of measuring the elastic response of live cells at specific locations in a physiologically friendly environment, with resolution of a few piconewton in force and a few tens nanometer in space [M. Pellegrino et al., Pflugers Arch – Eur J Physiol (2012) 464: 307–316.]. This is achieved by locally applying force stimuli by means of an external overpressure. Several kinds of live cells have been studied by SICM [J. Rheinlaender et al., Soft Matter (2013) 9: 3230-3236.].
The current experiment aims at measuring the spatial distribution of elasticity values in live cells in vitro using a SICM prototype developed on purpose in our lab. The experiment design is structured in subsequent steps:
1) development of a procedure to calibrate the size and the shape of the probe tip. This kind of information is necessary to evaluate the spatial resolution that can be achieved, the maximum cell slope that can be measured and other experimental parameters.
2) development of a procedure to calibrate the force applied to the sample surface in dependence on the probe-sample distance. Since SICM operates without direct contact between sample and probe, a proper working distance must be set, in order to optimize the transfer of the mechanical stimulus to the sample surface.
3) measurement of local elastic modulus of cells and correlation with the stiffness of their substrates. In fact, cells tune their internal stiffness to match that of their substrate, and modulation of the rigidity of cell environment may be used to direct cell migration and wound repair.
4) investigation of visco-elastic properties of cell. It has been observed, in fact, that the indentation depends on the force load rate, and thus the elastic modulus is also rate-dependent.
Measurement of indentation of different regions of a NIH 3T3 fibroblast. For details see ref. [1].
AFM image of the aperture of a 150 nm diameter pipette.