SLONANO2007- Ljubljana, October 10-12, 2007 1/22 PicoNewton Force Spectroscopy of Live Neuronal...
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Transcript of SLONANO2007- Ljubljana, October 10-12, 2007 1/22 PicoNewton Force Spectroscopy of Live Neuronal...
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: [email protected], http://www.tasc-infm.it
*CBM, Trieste; ^Univ. Magna Grecia, Catanzaro
SLONANO2007- Ljubljana, October 10-12, 2007 2/22
Outline
Motivation, goal, approach
Force spectroscopy using Optical Tweezers
Force measurements – Results
Conclusions
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
SLONANO2007- Ljubljana, October 10-12, 2007 4/22
Growth cone dynamics
Scale Bar = 5μmAcquisition freq= 0.2HzTime in min.sec
Movie available on request
Scale bar = 2 μm; Acquisition freq = 0.3Hz
Movie available on request
SLONANO2007- Ljubljana, October 10-12, 2007 5/22
Growth cones connection
Scale Bar = 3 μmAcquisition freq= 0.2HzTime in min.sec
Movie available on request
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
SLONANO2007- Ljubljana, October 10-12, 2007 7/22
Motivation, goal, approach
Force spectroscopy using Optical Tweezers
Force measurements – Results
Conclusions
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 setupIncluding force spectroscopy and multiple trapping
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)
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)
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
f0 – corner frequencyk – trap stifnessγ – Stokes drag coefficient of the bead
β – detector sensivityS0 – trap stifnessPV – plateu of
SLONANO2007- Ljubljana, October 10-12, 2007 12/22
Motivation, goal, approach
Force spectroscopy using Optical Tweezers
Force measurements – Results
Conclusions
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).
SLONANO2007- Ljubljana, October 10-12, 2007 14/22
Criteria to define a collision
Measurement away from Neuron
Measurement during collision
Measurements done byQPD & Video tracking Overlapped
SLONANO2007- Ljubljana, October 10-12, 2007 15/22
Results
Filopodia 2 minutes event, Fmax= 2pN
Movie available on request
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
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
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
SLONANO2007- Ljubljana, October 10-12, 2007 19/22
2 μm
Force exerted by Filopodia - Protrusion
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
SLONANO2007- Ljubljana, October 10-12, 2007 21/22
Multiple beads near Lamellipodia
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