[Show abstract][Hide abstract] ABSTRACT: Direct Laser Writing (DLW) is an innovative tool that allows the photo-fabrication of high resolution 3D structures, that can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of sub-micrometric patterned surfaces fabricated via DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 µm spaced sub-micrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 µm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the sub-micrometric patterned. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as sub-micrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology.
[Show abstract][Hide abstract] ABSTRACT: Scanning ion conductance microscopy (SICM) is a type of scanning probe microscopy based on the continuous measurement of an ion current flowing through a pipette filled with an electrolyte solution, while the pipette apex approaches a non-conductive sample. This technique can be operated in environmental conditions such as those of cell cultures and does not require a direct contact between probe and sample. It is therefore particularly suitable for the investigation of living specimens. SICM was initially proposed as an instrument that could obtain topographic 3D images with high resolution. Later, simple modifications have been devised to apply a mechanical stimulus to the specimen via a solution flux coming out from the pipette aperture. This modified setup has been employed to measure cell membrane elasticity and to guide the growth cones of neurons for tens of micrometers, by means of repeated non-contact scanning. Both these applications require an accurate measurement of the mechanical forces acting on the cell surface, which can be obtained by combining SICM, Atomic force microscopy (AFM) and inverted optical microscopy in the same apparatus. In this configuration, a SICM pipette is approached to an AFM cantilever while monitoring the cantilever deflection as a function of the pressure applied to the pipette and the relative distance. In addition, the pipette aperture can be imaged in situ by exploiting the AFM operation, so that all the experimental parameters can be effectively controlled in the investigation of pressure effects on living cells.
[Show abstract][Hide abstract] ABSTRACT: Knowledge of mechanical properties of living cells is essential to understand their physiological and pathological conditions. To measure local cellular elasticity, scanning probe techniques have been increasingly employed. In particular, non-contact scanning ion conductance microscopy (SICM) has been used for this purpose; thanks to the application of a hydrostatic pressure via the SICM pipette. However, the measurement of sample deformations induced by weak pressures at a short distance has not yet been carried out. A direct quantification of the applied pressure has not been also achieved up to now. These two issues are highly relevant, especially when one addresses the investigation of thin cell regions. In this paper, we present an approach to solve these problems based on the use of a setup integrating SICM, atomic force microscopy, and optical microscopy. In particular, we describe how we can directly image the pipette aperture in situ. Additionally, we can measure the force induced by a constant hydrostatic pressure applied via the pipette over the entire probe-sample distance range from a remote point to contact. Then, we demonstrate that the sample deformation induced by an external pressure applied to the pipette can be indirectly and reliably evaluated from the analysis of the current-displacement curves. This method allows us to measure the linear relationship between indentation and applied pressure on uniformly deformable elastomers of known Young's modulus. Finally, we apply the method to murine fibroblasts and we show that it is sensitive to local and temporally induced variations of the cell surface elasticity.
Pflügers Archiv - European Journal of Physiology 06/2012; 464(3):307-16. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Scanning ion conductance microscopy (SICM) is a scanning probe microscopy particularly suitable for the investigation of living biological specimens due to its low invasivity. Recently, this technique has been used not only to perform 3D-imaging, but also to stimulate and guide neuronal growth cones. In particular, it has been demonstrated that one can guide the cone growth for tens of micrometres by means of recurrent and non-contact SICM scanning along a defined line, with a pipette having an internal hydrostatic pressure. Accurate measurements of the mechanical forces acting on the cell membrane in these stimulation protocols are essential to explain the biological mechanisms involved. Herein a setup specifically developed for this purpose, combining together SICM, atomic force microscopy (AFM) and inverted optical microscopy is described. In this configuration, a SICM pipette can be approached to an AFM cantilever while monitoring the cantilever deflection as a function of the hydrostatic pressure applied to the pipette and the relative distance. In this way, one can directly measure mechanical forces down to 20 pN. The same apparatus is thus sufficient to calibrate a given pipette and immediately use it to study the hydrostatic pressure effects on living cells.
[Show abstract][Hide abstract] ABSTRACT: Scanning ion conductance microscopy (SICM) is currently used for high resolution topographic imaging of living cells. Recently, it has been also employed as a tool to deliver stimuli to the cells. In this work we have investigated the mechanical interaction occurring between the pipette tip and the sample during SICM operation. For the purpose, we have built a setup combining SICM with atomic force microscopy (AFM), where the AFM cantilever replaces the sample. Our data indicate that, operating in far-scanning mode with current decrease values below 2%, no force can be detected, provided that the level of the electrolyte filling the pipette is equal to that determined by the capillary tension. A filling level different from this value determines a hydrostatic pressure, a flux through the pipette tip and detectable forces, even in far-scanning mode. The absolute value of these forces depends on the pipette tip size. Therefore, a possible pitfall when using SICM for cell imaging is to imply zero-force working conditions. However the hydrostatic forces can be exploited in order to deliver weak mechanical stimuli and guide neuronal growth cones. Evidences of the effectiveness of this approach are herein given.
Neuroscience Research 03/2011; 69(3):234-40. · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We discuss on the feasibility of mapping the elastic properties of cellular regions with small thickness by means of a Scanning Ion Conductance Microscope employed to deliver mechanical stimuli in noncontact mode. I. INTRODUCTION In Scanning Ion Conductance Microscopy (SICM), a pipette is used as a probe to study the 3D morphology of cultured living cells. An ion current is let to flow through the pipette tip and the distance between probe and sample can be controlled by means of a feedback on the current signal. Upon properly choosing the working distance, topographic images of a living cell can be obtained with high resolution and without perturbing it.
[Show abstract][Hide abstract] ABSTRACT: Scanning ion conductance microscopy has been applied to neuronal growth cones of the leech either to image or to stimulate them. Growth cone advance was recorded in non-contact mode using a 2% ion current decrease criterion for pipette-membrane distance control. We demonstrate effective growth cone remodelling using a 5% criterion (near-scanning). Recurrent line near-scanning aligned growth cone processes along the scan line. The new membrane protrusions, marked by DiI, started a few minutes after scanning onset and progressively grew in thickness. Using scanning patterns suitable for connecting distinct growth cones, new links were consistently developed. Although the underlying mechanism is still a matter for investigation, a mechanical perturbation produced by the moving probe appeared to induce the process formation. Thanks to its deterministic and interactive features, this novel approach to guiding growth cones is a promising way to develop networks of identified neurons as well as link them with artificial structures.
Neuroscience Research 08/2009; 64(3):290-6. · 2.15 Impact Factor