Characterization of tip size and geometry of the pipettes used in scanning ion conductance microscopy.

Scanning ion-conductance microscopy (SICM) belongs to the family of scanning-probe microscopies. The spatial resolution of these techniques is limited by the size of the probe. In SICM the probe is a pipette, obtained by heating and pulling a glass capillary tubing. The size of the pipette tip is therefore an important parameter in SICM experiments. However, the characterization of the tip is not a consolidated routine in SICM experimental practice. In addition, potential and limitations of the different methods available for this characterization may not be known to all users. We present an overview of different methods for characterizing size and geometry of the pipette tip, with the aim of collecting and facilitating the use of several pieces of information appeared in the literature in a wide interval of time under different disciplines. In fact, several methods that have been developed for pipettes used in cell physiology can be also fruitfully employed in the characterization of the SICM probes. The overview includes imaging techniques, such as scanning electron microscopy and atomic Force microscopy, and indirect methods, which measure some physical parameter related to the size of the pipette. Examples of these parameters are the electrical resistance of the pipette filled with a saline solution and the surface tension at the pipette tip. We discuss advantages and drawbacks of the methods, which may be helpful in answering a wide range of experimental questions.

Comment on "The long range voice coil atomic force microscope" [Rev. Sci. Instrum. 83, 023705 (2012)].

In a recent article Barnard et al. described the use of voice coil actuators to realize a large range scanner for probe microscopy. The results reported are interesting, but the idea is not new. In two preceding papers [1998, 1991] we had described a large coverage, wide dynamic range scanner based on homemade voice coil actuators, while Garcia Cantu and Huerta Garnica [1986] had already used inductive scanners for tunneling microscopy. Lamentably, none of these articles was cited by Barnard et al.

Measuring the elastic properties of living cells through the analysis of current-displacement curves in scanning ion conductance microscopy.

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.

Weak hydrostatic forces in far-scanning ion conductance microscopy used to guide neuronal growth cones.

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.

Recapitulation of the Roberts syndrome cellular phenotype by inhibition of INCENP, ZWINT-1 and ZW10 genes.

Roberts syndrome is an autosomal recessive disorder characterised primarily by symmetric reduction of all limbs and growth retardation. Patients have been reported to have premature separation of heterochromatin regions of many chromosomes and abnormalities in cell cycle. Given the rarity of the syndrome, the linkage analysis approach is not suitable to identify the responsible gene. In this work, a cell line derived from a patient affected by Roberts syndrome was characterized by cell biology and molecular cytogenetics, including comparative genomic hybridization and spectral karyotype. No recurrent chromosomal rearrangements were identified. Thereafter, based on the fact that premature chromatide separation is a reliable marker of the disease, we used antisense oligonucleotide technologies to inhibit six genes involved in various steps of the correct chromosome segregation, such as chromosome cohesion, kinetochore assembling, spindle checkpoint and spindle formation. We found that the inhibition of INCENP, ZWINT-1, ZW10 genes results in the appearance of mitotic cells characterised by centromere separation, chromosome aneuploidy and micronuclei formation. In addition, INCENP, ZWINT-1, ZW10 antisense-treated chromosome morphology was very similar to that of Roberts chromosome when analysed by atomic force microscopy. We concluded that INCENP, ZWINT-1, ZW10 gene inhibition results in cellular phenocopies of Roberts syndrome. Taken together, these findings support a possible role of these genes in the pathogenesis of Roberts syndrome.