Magnetic domain wall tweezers: a new tool for mechanobiology studies on individual target cells.

In vitro tests are of fundamental importance for investigating cell mechanisms in response to mechanical stimuli or the impact of the genotype on cell mechanical properties. In particular, the application of controlled forces to activate specific bio-pathways and investigate their effects, mimicking the role of the cellular environment, is becoming a prominent approach in the emerging field of mechanobiology. Here, we present an on-chip device based on magnetic domain wall manipulators, which allows the application of finely controlled and localized forces on target living cells. In particular, we demonstrate the application of a magnetic force in the order of hundreds of pN on the membrane of HeLa cells cultured on-chip, via manipulation of 1 µm superparamagnetic beads. Such a mechanical stimulus produces a sizable local indentation of the cellular membrane of about 2 µm. Upon evaluation of the beads' position within the magnetic field originated by the domain wall, the force applied during the experiments is accurately quantified via micromagnetic simulations. The obtained value is in good agreement with that calculated by the application of an elastic model to the cellular membrane.

Photoactivation of pa-GFP in 3D: optical tools for spatial confinement.

Photoactivatable fluorescent proteins represent an innovative tool for the direct observation of time dependent macromolecular events in living systems. The possibility of switching on a selected and confined subset of the expressed target proteins allows to follow biological processes reaching high signal to noise ratios. In particular, use of non-linear interactions to bring the molecules in the activated fluorescent form make it possible to extend the advantages of photoactivation to events that requires 3D spatial localization. In this work, we show the possibility to realize confined activated volumes in living cells, by employing photoactivatable green fluorescent protein (paGFP) in two-photon microscopy. The analysis of the kinetics of two-photon paGFP activation in dependence of the wavelength, the laser intensity and the exposure time is provided. This study allowed to assess the optimal conditions to induce photoactivation in living samples and to track the behaviour of tagged histone H2B during cellular division. Furthermore we investigate paGFP photoactivation under evanescent wave illumination. Total internal reflection set-up has been used to selectively activate subresolved distribution of proteins localized in the basal membrane surroundings. These two photoactivation methods provide a suitable tool for many biological applications, combining subresolved surface and in-depth three-dimensionally confined investigations.

Spatial control of pa-GFP photoactivation in living cells.

Photoactivatable green fluorescent protein (paGFP) exhibits peculiar photo-physical properties making it an invaluable tool for protein/cell tracking in living cells/organisms. paGFP is normally excited in the violet range (405 nm), with an emission peak centred at 520 nm. Absorption cross-section at 488 nm is low in the not-activated form. However, when irradiated with high-energy fluxes at 405 nm, the protein shows a dramatic change in its absorption spectra becoming efficiently excitable at 488 nm. Confocal microscopes allow to control activation in the focal plane. Unfortunately, irradiation extends to the entire illumination volume, making impracticable to limit the process in the 3D (three-dimensional) space. In order to confine the process, we used two advanced intrinsically 3D confined optical methods, namely: total internal reflection fluorescence (TIRF) and two-photon excitation fluorescence (2PE) microscopy. TIRF allows for spatially selected excitation of fluorescent molecules within a thin region at interfaces, i.e. cellular membranes. Optimization of the TIRF optical set-up allowed us to demonstrate photoactivation of paGFP fused to different membrane localizing proteins. Exploitation of the penetration depth showed that activation is efficiently 3D confined even if limited at the interface. 2PE microscopy overcomes both the extended excitation volume of the confocal case and the TIRF constraint of operating at interfaces, providing optical confinement at any focal plane in the specimen within subfemtoliter volumes. The presented results emphasize how photoactivation by non-linear excitation can provide a tool to increase contrast in widefield and confocal cellular imaging.