Atomic Force Microscopy ISC-CNR

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1 Atomic Force Microscopy ISC-CNR ICS-CNRCNR Bruno Tiribilli Imaging on Biological samples Thin section of tissue Protein aggregates Force measurement Cells elasticity Protein unfolding Design and realization Mechanics Control logic Optical excitation of cantilever

2 AFM topography AFM topography of C2C12 cell contact mode with MI PicoSPM; 60 m x 60 m

3 Imaging Thin Section of Biological Tissue B.Tiribilli, D.Bani, F.Quercioli, A.Ghirelli, M.Vassalli, "Atomic force microscopy of histological sections using a chemical etching method". Ultramicroscopy 102, (2005)

4 AFM imaging on tissue Equipment costs K Resolution comparable to TEM Sample preparation time The whole surface is available for imaging (no grid) Sample can be simultaneously observed by optical microscope

5 Imaging Protein aggregates Cerato platanin is a phytotoxic protein of 12.4 kda released by the Ascomycete fungus Ceratocystis fimbriata f. sp. platani, responsible of the canker stain disease that affects Platanus acerifolia.

6 Imaging with Pulse Force Mode Figura Witec A -TOPOGRAPHY B -DEFLECTION C - STIFFNESS cell C2C12 area = 25 m x 25 m

7 Elasticity of Cells The modified cantilever can be used as a nanoindenter to measure mechanical parameters of biological samples ROLE FOR MYOSIN II AND STRESS FIBER CONTRACTION ON MEMBRANE TENSION DEVELOPMENT AND STRETCH-ACTIVATED CHANNEL (SAC) ACTIVATION. ( F. Sbrana, C.Sassoli, D. Nosi, R. Squecco, F. Paternostro, B.Tiribilli, S. Zecchi-Orlandini, F. Francini, L. Formigli.

8 Single Molecule Stretching Experiment The tip stay in contact and redraw Resistance at the extension, the force rises A domain begins to unfolds The force increase until the protein unfold completely The force drops Stretching experiments on polymeric protein result in force-distance curves showing a characteristic sawtooth pattern The peaks of the sawtooth pattern correspond to the consecutive mechanical unfolding of individual domains

9 Atomic Force Microscope SPMagic controller with analog PI feedback Linearized piezo scanner 80 µm x80 µm x10µm Contact and Tapping in air

10 AFM for liquid environment

11 Recent publications ICS-CNRCNR F. Sbrana, D. Fanelli, M. Vassalli, L. Carresi, A. Scala, L. Pazzagli, G. Cappugi, Bruno Tiribilli Progressive pearl necklace collapse mechanism for cerato-ulmin aggregation film Eur Biophys J DOI /s (2009) C. Comparini, L. Carresi, E. Pagni, F. Sbrana, F Sebastiani, N. Luchi, A. Santini, P. Capretti, B. Tiribilli, L. Pazzagli, G. Cappugi, A. Scala. New Proteins orthologous to cerato-platanin in various Ceratocystis species and the purificaation and characterization of the cerato populin from Ceratocistis populonica. Appl Microbiol Biotechnol DOI /s (2009) P. Matteini, F. Sbrana, B. Tiribilli, R. Pini Low temperature corneal laser welding investigated by atomic force microscopy Ophthalmic Technologies XIX. Proceedings of the SPIE, Volume 7163, pp Q Q-5 (2009). S. Marchetti, F. Sbrana, R. Raccis, L. Lanzi, C.M.C. Gambi, M.Vassalli, B.Tiribilli, A.Pacini, A.Toscano Dynamic light scattering and atomic force microscopy imaging on fragments of β-connectin from human cardiac muscle, PRE, 77, (2008) F. Sbrana, C. Sassoli, E. Meacci, D. Nosi, R. Squecco, F. Paternostro, B. Tiribilli, S. Zecchi-Orlandini, F. Francini, L. Formigli, Role for stress fiber contraction in surface tension development and stretchactivated channel regulation in C2C12 myoblasts Am J Physiol Cell Physiol., vol. 295, pp. C160 - C172 (2008) P. Matteini, F. Sbrana, B. Tiribilli, R. Pini Atomic force microscopy and transmission electron microscopy analyses of low-temperature laser welding of the cornea Lase Med. Sci. DOI /s (2008) M. Basso, P. Paoletti, B. Tiribilli, M. Vassalli Modelling and analysis of autonomous micro-cantilever oscillations, Nanotechnology, 19, (2008)