The morphology and the nanomechanical properties of single casein micelles (CMs) in their native state were investigated using AFM in liquid environment. A novel immobilization method of CMs using low energy interactions was developed and compared with a well-known chemical immobilization method involving direct covalent bonds.For the classic method, CMs were covalently bound via carbodiimide chemistry to a gold-sputtered glass surface precoated with carboxyl groups. For the proposed method an indirect immobilization of CMs was realized via anti-human phospho-Ser/Thr/Tyr mouse monoclonal antibody covalently bound onto a gold-coated slide via carbodiimide chemistry. In both methods, immobilized CMs were then analyzed by AFM in their native environment represented by a simulated milk ultrafiltrate at room temperature.With the classic method of immobilization, some CMs remained mobile in liquid condition during the probe sample interaction. Conversely, with the novel method, CMs were well immobilized allowing the extraction of both surface topography and elastic properties by AFM in liquid. Morphologic characteristics were obtained from 2D and 3D images (Figure1). Data analyses allowed the calculation of diameters, heights and contact angles of native CMs. They are characterized by a mean width of 148 ± 8 nm and a mean height of 42 ± 1 nm. Low forces were applied on immobilized CM to calculate force-vs-indentation curves fitted using Hertz theory in order to determine Young Moduli (E*) which represents elasticity. Their elasticity distribution exhibited a unimodal trend, with an elasticity pic centered at 269 ± 14 kPa.

Peripheral nerve injuries are serious traumas resulting in loss of motor and sensory functions of nerves. The post-traumatic neuropathies are often chronic and mostly resistant to current treatments. Our objective is to develop new strategies to regenerate nerve after trauma by biomimetic approaches and go towards in vitro neurogenesis. For this we use neural crest derived human multipotent dental pulp stem cells (hDPSC) in co-culture with sensory neurons from mice dorsal root ganglia (DRG), and an in vitro neurogenic differentiation of hDPSC [3].In order to explore nanomechanical and morphological characteristics of human DPSC and mouse DRG neurons atomic force microscopy was employed. We have shown that the mechanical properties of the soma and sub cellular growth cone regions correlate well with the amount, organization and dynamics of the underlying cytoskeletal structure [1,2]. Here we present preliminary data about hDPSC in co-culture, with mDRG neurons and during an in vitro neurogenic differentiation gathered by nanomechanical mapping.[1] O. Benzina, V. Szabo, O. Lucas, M-b. Saab, T. Cloitre, F. Scamps, C. Gergely and M. Martin (2013) Changes induced by peripheral nerve injury in the morphology and nanomechanics of sensory neurons, J. Biomedical Optics 18(10)[2] M. Martin, O.Benzina, V. Szabó, A. G. Végh, O. Lucas, T. Cloitre, F. Scamps and C. Gergely (2013) Morphology and Nanomechanics of Sensory Neurons Growth Cones following Peripheral Nerve Injury, Plos One , 8 (2)[3] M. Király, B. Porcsalmy, A. Pataki, K. Kádár, M. Jelitai, B. Molnár, P. Hermann, I. Gera, WD. Grimm, B. Ganss, A. Zsembery, G. Varga, (2009) Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons, Neurochem. Int. 55 (323-332)

Fluorescent nano-diamonds (fND) are attractive tools for nanoscale biological cellular imaging allowing both photoluminescence and magnetic resonance imaging [1]. Recent technological developments enable to fabricate bright fND particles of various sizes with high content of nitrogen-vacancy (NV) centres [2]. In this work we study internalization processes of NDs in the breast cancer MCF7 1DIV cell line using Atomic Force Microscopy imaging and force spectroscopy. fND particles of size range from 5 to 60 nm were used, prepared from Ib synthetic diamond, electron irradiated, annealed and plasma oxidized to create NV centers.Changes of cells stiffness were detected by AFM force measurements after introducing the fNDs into the cell medium. We observed an oscillating variation of cell membrane Young's modulus while the cells become stiffer. We believe that repetitive uptake/release processes of fNDs are responsible for these mechanical changes. Contrarywise, more confluent MCF7 cells (3DIV) did not show any significant change in Young’s Modulus, as compare with control ones. Indeed, it has been shown that differences in nanoparticle uptake can arise from how close the cells are to one another (confluence) [3] and how old they are.Moreover, our results suggest that studies on fNDs uptake should consider the cell cycle, as in a cell population, the dose of internalized nanoparticles in each cell varies as the expression of membrane proteins vary during the cell cycle [4]. The cell cycle is a series of events that lead to cell division and replication, consisting of four phases: G1 (when the cell increases its size), S (the cell synthesizes DNA), G2 (the cell synthesizes proteins for cell division) and M (the cell divides and the two daughter cells enter the G1 phase). During each event, cellular processes can vary; meaning that the rate at which a cell takes up foreign material, as for instance nanoparticles, may depend on the phase the cell is in [4-6], modifying accordingly their elasticity. When nanoparticle internalization is studied, it is therefore crucial to resolve how the state of the different cells affects the uptake. [1] L. Moore, M. Nesladek et al., Nanoscale (2014).[2] J. Havlik, M. Gulka, M. Nesladek et al., Nanoscale (2013).[3] B. Snijder, et al. Nature 461, 520–523 (2009). [4] E. Boucrot and T. Kirchhausen, Proc. Natl Acad. Sci. USA 104, 7939–7944 (2007). [5] D. Raucher and M. P. Sheetz, J. Cell Biol. 144, 497–506 (1999). 
[6] J. K. Schweitzer, E. E. Burke, H. V Goodson and C. D’Souza-Schorey. J. Biol. Chem. 280, 41628–41635 (2005).