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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

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Outline

Motivation, goal, approach

Force spectroscopy using Optical Tweezers

Force measurements – Results

Conclusions

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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

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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

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Growth cones connection

Scale Bar = 3 μmAcquisition freq= 0.2HzTime in min.sec

Movie available on request

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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

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Motivation, goal, approach

Force spectroscopy using Optical Tweezers

Force measurements – Results

Conclusions

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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

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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)

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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)

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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

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Motivation, goal, approach

Force spectroscopy using Optical Tweezers

Force measurements – Results

Conclusions

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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).

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Criteria to define a collision

Measurement away from Neuron

Measurement during collision

Measurements done byQPD & Video tracking Overlapped

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Results

Filopodia 2 minutes event, Fmax= 2pN

Movie available on request

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Results

Lamellipodia 2 minutes event, F> 20pN

Clicking on !! You might see the lamellipodia

taking the bead out from the trap

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Force exerted by Lamellipodia

Acquisition rate: 20Hz

Scale Bar = 2μm

Time in seconds

Acquisition rate : 4KHz

Subsampeled at : 2KHz

Movie available on request

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Force exerted by Filopodia - Protrusion

Acquisition rate: 20Hz

Scale Bar = 2μm

Time in seconds

Acquisition rate : 4KHz

Subsampeled at : 2KHz

Movie available on request

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2 μm

Force exerted by Filopodia - Protrusion

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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

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Multiple beads near Lamellipodia

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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