We have built a fully interactive three-dimensional (3-D) nanomanipulator integrated with an optical trapping-based Photonic Force Microscope (PFM) for in-vivo and in-situ operation inside living cells. A PFM is combined with a 3-D real-time haptic (force feedback) nanomanipulator. The system is based on an optical trap with an ultra-fast 3-D position detection system. It allows to manipulate in 3-D a probe inserted inside a living cell and to record its position with nanometer spatial and microsecond temporal resolution. By mapping the thermally induced movements of the probe, it is possible to measure intracellular forces and the 3-D stiffness matrix in real time. This instrument will allow interactive accessing, positioning, measuring, imaging, and reacting.
Was ist das Besondere an diesem Projekt?
The traditional approach in PFM studies, by application of suitable statistical models, brings valuable data concerning the investigated medium. Successful determination of viscoelastic properties of aqueous medium, mechanical properties of molecules, or 3D topology of polymer network has thus been reported. This technique however requires extensive, off-line analysis of huge datasets - the interactive reaction of the scientist conducting the experiment is not possible. Our system through the use of dedicated analog signal processor and the dual data acquisition, introduces the real-time performance into the PFM experiment, preserving simultaneously all benefits of the traditional approach.
By combining the PFM with the haptic nanomanipulator, we made possible to pick, place, and release the probe in a minimally invasive way. The haptic 3-D force feedback mechanism allows the operator to feel and react online with a joystick to changes (e.g. in forces, local viscosity, sample stiffness) in the probe's surrounding environment.
In order to cope with a high data throughput we have built an analog processor, extracting in real time the 3-D stiffness data. Such a device can be seen as a nano-bio-laboratory where extensive information concerning the sampled volume is available in real time and the probe can be interactively positioned inside a cell.
The protocol of introducing 240 nm polystyrene beads inside a cell was elaborated. It enables to place beads in the cytoplasm within 12 hours of incubation of cells with the solution containing few probing beads. The incubation time is dependent on the bead size, surface charge, coating. Currently the way of introducing the beads into cell nucleus, using electric field, is under development. The identification of beads inside cells is realized by the fluorescence optical chain that was added to the system.
The chosen biological model for probing cell interior was based on two cell lines from human bladder: non-malignant cells of urether (HCV29 cell line) and malignant bladder cells (T24 cell lines). Together with PFM measurements, the characterization of cell interior has been performed using TEM (transmission electron microscope) and fluorescence microscope in order to get information on the spatial distribution of a cell cytoskeleton and cytoplasm within cell interior.
The first results of cell interior probing showed similar values of stiffness inside cells but the character of its distribution was different depending on the cell type. To characterize the differences in a quantitative way, the parameters describing the stiffness anisotrophy has been delivered. It showed that the probing beads have more space to fluctuate in canceorus cells while in non-malignant cells the beads fluctuates along a defined direction.
Project resulted with three published papers and two conference contributions. The fourth paper is under preparation.
Sylvia Jeney, Branimir Lukic, Jonas A. Kraus, Thomas Franosch, and Laszlo Forro: Anisotropic Memory Effects in Confined Colloidal Diffusion, Phys. Rev. Lett. 100, 240604 (2008)
Camilo Guzman, Henrik Flyvbjerg, Roland Köszali, Carole Ecoffet, Laszlo Forro, and Sylvia Jeney: In situ viscometry by optical trapping interferometry, Appl. Phys. Lett. 93, 184102 (2008)
E Bertseva, A S G Singh, J Lekki, P Thevenaz, M Lekka, S Jeney, G Gremaud, S Puttini, W Nowak, G Dietler, L Forro, M Unser and A J Kulik: Intracellular nanomanipulation by a photonic-force microscope with real-time acquisition of a 3D stiffness matrix, Nanotechnology 20 (2009) 285709 (9pp)
Abstract: A.J. Kulik, M. Lekka, J. Lekki, E. Bertseva, P. Thévenaz, A. Singh, S. Jeney, M. Unser, L. Forro, "Intracellular Manipulation Using Optical Tweezers," Tenth School on Acousto-Optics and Applications (AOA'08), Gdansk-Sopot, Poland, May 12-15, 2008
Abstract: A.J. Kulik, J. Lekki, , M. Lekka, E. Bertseva, A. Singh, P. Thévenaz, S. Puttini, S. Jeney, W. Nowak, G. Dietler, M. Unser, L. Forro, "Intracellular 3-D Interactive Nanomanipulator" , Eleventh Annual Linz Winter Workshop: Advances in Single-Molecule Research for Biology & Science, Linz, Austria, 6-10 Feb 2009
P. Thévenaz, A. S. G. Singh, E. Bertseva, J. Lekki, A. J. Kulik, M. Unser: Model-Based Estimation of 3-D Stiffness Parameters in Photonic-Force Microscopy, IEEE Transactions on Nanoscience, vol. 9, no. 2, June 2010
Am Projekt beteiligte Personen
Prof M. Unser, Ph. Thévenaz, EPFL-BIG, Biomedica Imaging Group, Lausanne
Prof G. Dietler, S. Kasas EPFL-LPMV, Laboratory of Physics of Living Matter, Lausanne
Drs M. Lekka, J. Lekki, INP-PAN, Polish Academy of Scineces, Institute of Nuclear Physics, Krakow, Poland
Prof W. Nowak, UMK, Nicholas Copernicus University, Institute of Physics, Torun, Poland
Letzte Aktualisierung dieser Projektdarstellung 24.10.2018