1. SLONANO2007- Ljubljana, October 10-12, 2007 1/22 PicoNewton Force Spectroscopy of Live Neuronal Cells using Optical Tweezers * Dan Cojoc, Enrico Ferrari, Francesco Di Fato, Rajesh Shahapure, Jumi Laishram, Massimo Righini, ^Enzo Di Fabrizio, Vincent Torre CNR – INFM, Laboratorio Nazionale TASC, Trieste SISSA, Neurobiology sector, Trieste E-mail: cojoc@tasc.infm.it, http://www.tasc-infm.it *CBM, Trieste; ^Univ. Magna Grecia, Catanzaro

2. SLONANO2007- Ljubljana, October 10-12, 2007 2/22 Outline  Motivation, goal, approach  Force spectroscopy using Optical Tweezers  Force measurements – Results  Conclusions

3. SLONANO2007- Ljubljana, October 10-12, 2007 3/22 www.biology.lsa.umich.edu/research/labs/ktosney / Motivation and goal of our work Structural elements of the growth cone Key determinant of axonal growth is the growth cone: "They will adopt pre-determined directions and establish connections with defined neural or extra neural elements... without deviations or errors, as if guided by an intelligent force." 1890 RAMON Y CAJAL

4. SLONANO2007- Ljubljana, October 10-12, 2007 4/22 Growth cone dynamics Scale Bar = 5μm Acquisition freq= 0.2Hz Time in min.sec Movie available on request Scale bar = 2 μm; Acquisition freq = 0.3Hz Movie available on request

5. SLONANO2007- Ljubljana, October 10-12, 2007 5/22 Growth cones connection Scale Bar = 3 μm Acquisition freq= 0.2Hz Time in min.sec Movie available on request

6. SLONANO2007- Ljubljana, October 10-12, 2007 6/22 J.L. Goldberg, Genes and Dev. 17 941 (2003) Goal - Approach -Calibrate the trap by measuring the fluctuations of the bead in trap -Micro beads trapped by IR laser and positioned in front of lamellipodia and/or filopodia -Measure the fluctuations of the bead in the trap, due to its interaction with the neurite, and convert them into forces. Experimental approach Goal: measure the forces exerted by lamellipodia and filopodia

7. SLONANO2007- Ljubljana, October 10-12, 2007 7/22  Motivation, goal, approach  Force spectroscopy using Optical Tweezers  Force measurements – Results  Conclusions

8. SLONANO2007- Ljubljana, October 10-12, 2007 8/22 Bead position was determined by back focal plane (BFP) detection: BFP of the condenser was imaged onto a QPD Force = K. Δ X K = stiffness of the trap (spring constant) ΔX = Displacement Optical Tweezers setup Including force spectroscopy and multiple trapping

9. SLONANO2007- Ljubljana, October 10-12, 2007 9/22 Schematic of a μm bead diffusing in an optical trap Mechanical model of the forces acting on the bead Trap calibration from the fluctuations of the bead The power spectrum density S ( f ) of these fluctuations near the center of an optical trap is approximately Lorentzian (Svoboda and Block, 1994; Gittes and Schmidt, 1997)

10. SLONANO2007- Ljubljana, October 10-12, 2007 10/22 Back focal plane interferometry detect the thermal fluctuations of the bead with Displacement from the focus Centered XY Z Voltage change on the detector F. Gittes, Optics Letters, (1998)

11. SLONANO2007- Ljubljana, October 10-12, 2007 11/22 Trap stiffness and detector sensivity The power spectrum (dotted line) of a trapped 1 μm silica bead acquired at 10 KHz and fitted to a Lorentzian (solid line). Sv(f) - measured power spectrum S(f) - density Lorentzian fit f 0 – corner frequency k – trap stifness γ – Stokes drag coefficient of the bead β – detector sensivity S 0 – trap stifness PV – plateu of

12. SLONANO2007- Ljubljana, October 10-12, 2007 12/22  Motivation, goal, approach  Force spectroscopy using Optical Tweezers  Force measurements – Results  Conclusions

13. SLONANO2007- Ljubljana, October 10-12, 2007 13/22 Features of our setup Trap stiffness: 5-100 pN/μm Resolution: ~10nm (1 nm) Force range: 1-25 pN Errors are about 10% (Some) Problems encountered: Stuck beads to the substrate Trapping and calibration close to the substrate (<2 μm ) and at T=37 C Influence of floating particles on the interference pattern Filopodia collisions reveal lower forces than expected ? Tam-Tam ! Experimental results Neurons obtained from dorsal root ganglia (DRG), isolated from P0-12 rats and plated on poly-L-lysine-coated glass dishes. 48 hours after incubation in 50 ng/ml of nerve growth factor (NGF).

14. SLONANO2007- Ljubljana, October 10-12, 2007 14/22 Criteria to define a collision Measurement away from Neuron Measurement during collision Measurements done by QPD & Video tracking Overlapped

15. SLONANO2007- Ljubljana, October 10-12, 2007 15/22 Results Filopodia 2 minutes event, Fmax= 2pN Movie available on request

16. SLONANO2007- Ljubljana, October 10-12, 2007 16/22 Results Lamellipodia 2 minutes event, F> 20pN Clicking on !! You might see the lamellipodia taking the bead out from the trap

17. SLONANO2007- Ljubljana, October 10-12, 2007 17/22 Force exerted by Lamellipodia Acquisition rate: 20Hz Scale Bar = 2μm Time in seconds Acquisition rate : 4KHz Subsampeled at : 2KHz Movie available on request

18. SLONANO2007- Ljubljana, October 10-12, 2007 18/22 Force exerted by Filopodia - Protrusion Acquisition rate: 20Hz Scale Bar = 2μm Time in seconds Acquisition rate : 4KHz Subsampeled at : 2KHz Movie available on request

19. SLONANO2007- Ljubljana, October 10-12, 2007 19/22 2 μm Force exerted by Filopodia - Protrusion

20. SLONANO2007- Ljubljana, October 10-12, 2007 20/22 Force exerted by Filopodia - Lateral collision Acquisition rate: 20Hz Scale Bar = 2μm Numbers indicate time in seconds Acquisition rate : 4KHz Subsampeled at : 2KHz Movie available on request

21. SLONANO2007- Ljubljana, October 10-12, 2007 21/22 Multiple beads near Lamellipodia

22. SLONANO2007- Ljubljana, October 10-12, 2007 22/22 Conclusions  Introduce a method to measure pN forces expressed by filopodia (3 pN) and lamellipodia (more than 20 pN)   PlosOne accepted Sept 2007  Found that even one neuron is (very) intelligent  Found lot of questions to answer to in the future work Acknowledgments TASC : Enrico Ferrari, Valeria Garbin, Lilit Group SISSA : Vincent Torre, Rajesh Shahapure, Massimo Righi Francesco Difato, Jummi Laishram