NER: NANO-GRATING FORCE SENSOR FOR MEASUREMENT OF NEURON MEMBRANE CHARACTERISTICS UNDER GROWTH AND CELLULAR DIFFERENTIATION Ashwini Gopal 1 , Zhiquan Luo 2 , Karthik Kumar 1 , Jae Young Lee 3 , Kazunori Hoshino 1 , Bin Li 2 , Christine Schmidt 1, 3 , Paul S. Ho 2 , and Xiaojing Zhang 1 Departments of Biomedical Engineering 1 , Mechanical Engineering 2 , Chemical Engineering 3 The University of Texas at Austin,Texas, USA •Cell experiences stress and strain Physical/Chemical cues (growth) Environmental perturbation (robustness) •Neuronal shape depend on both progressive and regressive processes such as axonal elongation and axonal elimination [2-3] •Mechanical tension is a direct stimulus for neurite initiation and elongation from peripheral neurons Classical concepts fail to explain mechano-transduction [4] Size-dependant mechanical properties have not been characterized •PC12-a cell line derived from rat pheochromocytoma •Serve as good models of neuron cells •Easily be induced to extend neurites with NGF guided nerve regeneration wound healing cell based drug delivery Motivation Basic structure of neuron [6] PC12 Cells [5] Cell Structure [1] Principle of Operation 2 2 1 p m p m p •Sensitivity of the device can be greatly improved by reducing the critical feature size, p, of the grating to the nanometer regime to match the illumination wavelength. •Probe displacement, therefore the force, can be measured through the spatial frequency of far-field diffraction pattern. m is diffraction order,λ is wavelength, p is grating pitch, α is illumination angle, and θ is diffraction angle. sin sin p m Device Design •Two symmetric nanogratings provide mechanical balance and maximal optical surface area. •Nanogratings consist of flexure folding beams suspended between two known stiffness [7] . 3 3 2 s s L t Ew k Suspension Stiffness 3 3 8 f f L t Ew k Flexure Stiffness Simulation Results Eigen modes under mechanical vibration were simulated using CoventorWare™ . Absence of stress across the gratings and stress concentrated on the flexure beams Fabrication Sequence SEM Images Experimental Setup •Nanograting sensor is attached to a piezoelectric actuator •Placed in a liquid media (serum containing F12K media) to probe the PC12 cells (Neurons) •Illuminated with a 635 nm He-Ne laser diode •Spot size covering the grating area of 120μm x 120μm. •Experiment was conducted on 10-15 cells •Time for testing cells-30mins •An average force of 22+ 8μN caused the neurite length to contract up to 6μm Results Conclusions Opto-mechanical sensing interface to study cell mechanics •Design, simulation, and fabrication of nanograting displacement-force sensor •Displacement range of 10µm & force sensitivity 8N/m •Eigen modes vs. optical detection •Localized, quantitative interactions in microenvironment Neuron(PC12) mechanics characterization •Neurite contraction under mechanical stimulation •Elastic modulus of neuron membrane 425±30 Pa Platform to study mechano-transduction in other cell lines •Hippocampal cells, spinal cord motor neuron x: Experimental values * : Calibration Values •Probing causes motion of grating •Change in diffraction spot •Determines the amount of force applied. (F=kx) Reference: Reference: [1] See: http://www.uic.edu/classes/bios/bios100/lecturesf04am/cyt oskeleton.jpg [2] Cowan, W.M, Fawcett, J. W, O'Leary, D. D. M, and Stanfield, B. B “Regressive events in neumgenesis.” Science. 225, p1258-1265, 1984. [3] Purves, D, and Lichtman, J. W, “Elimination of synapses in the developing nervous system.”, Science, 210, p153- 157, 1980. [4] McKintosh F C, Kas J and Janmey P A 1995 Phys. Rev. Lett. 75 4425 [5] See: http://scienceblogs.com/purepedantry/2006/07/background_t o_the_20_year_coma.php [6] See: http://www.med.osaka-u.ac.jp/pub/molonc/www/Esignal.html [7] Stephen D. Sentura “ Microsystem Design”,2001 Acknowledgement: NSF Nanoscale Exploratory Research Award (NER), ECS-0609413, the Welch Foundation and the Strategic Partnership for Research in Nanotechnology (SPRING), and NSF NNIN Facilities at UT Austin and Stanford Center for Integrated Systems (Grant # 0335765 and 9731293 respectively)
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NER: NANO-GRATING FORCE SENSOR FOR MEASUREMENT OF NEURON MEMBRANE CHARACTERISTICS UNDER GROWTH AND CELLULAR DIFFERENTIATION
Ashwini Gopal1, Zhiquan Luo2, Karthik Kumar1, Jae Young Lee3, Kazunori Hoshino1, Bin Li2, Christine Schmidt1, 3, Paul S. Ho2, and Xiaojing Zhang1 Departments of Biomedical Engineering1, Mechanical Engineering2, Chemical Engineering3The University of Texas at Austin,Texas, USA
• Cell experiences stress and strainPhysical/Chemical cues (growth)Environmental perturbation (robustness)
• Neuronal shape depend on both progressive and regressive processes such as axonal elongation and axonal elimination [2-3]
• Mechanical tension is a direct stimulus for neurite initiation and elongation from peripheral neurons
Classical concepts fail to explain mechano-transduction[4]
Size-dependant mechanical properties have not been characterized
• PC12-a cell line derived from rat pheochromocytoma
•Serve as good models of neuron cells•Easily be induced to extend neurites with NGF
• Significanceguided nerve regenerationwound healing cell based drug delivery
Motivation
Basic structure of neuron [6]
PC12 Cells [5]
Cell Structure [1]
Principle of Operation
2
2 1
pm
p
m
p
•Sensitivity of the device can be greatly improved by reducing the critical feature size, p, of the grating to the nanometer regime to match the illumination wavelength.
•Probe displacement, therefore the force, can be measured through the spatial frequency of far-field diffraction pattern.
m is diffraction order,λ is wavelength, p is grating pitch, α is illumination angle, and θ is diffraction angle.
sinsin pm
Device Design
•Two symmetric nanogratings provide mechanical balance and maximal optical surface area.
•Nanogratings consist of flexure folding beams suspended between two parallel cantilevers of known stiffness[7].
3
32
ss
L
tEwk
Suspension Stiffness
3
38
ff
L
tEwk
Flexure Stiffness
Simulation Results
Eigen modes under mechanical vibration were simulated using CoventorWare™ .
Absence of stress across the gratings and stress concentrated on the flexure beams
Fabrication Sequence
SEM Images
Experimental Setup
• Nanograting sensor is attached to a piezoelectric actuator• Placed in a liquid media (serum containing F12K media) to probe the PC12 cells (Neurons)
• Illuminated with a 635 nm He-Ne laser diode• Spot size covering the grating area of 120μm x 120μm.
• Experiment was conducted on 10-15 cells • Time for testing cells-30mins• An average force of 22+8μN caused the neurite length to
contract up to 6μm
Results
Conclusions
Opto-mechanical sensing interface to study cell mechanics• Design, simulation, and fabrication of nanograting
displacement-force sensor• Displacement range of 10µm & force sensitivity 8N/m • Eigen modes vs. optical detection• Localized, quantitative interactions in microenvironment
Neuron(PC12) mechanics characterization• Neurite contraction under mechanical stimulation• Elastic modulus of neuron membrane 425±30 Pa
Platform to study mechano-transduction in other cell lines• Hippocampal cells, spinal cord motor neuron
x: Experimental values*: Calibration Values
•Probing causes motion of grating •Change in diffraction spot•Determines the amount of force applied. (F=kx)
Reference:Reference:
[1] See: http://www.uic.edu/classes/bios/bios100/lecturesf04am/cytoskeleton.jpg[2] Cowan, W.M, Fawcett, J. W, O'Leary, D. D. M, and Stanfield, B. B “Regressive
events in neumgenesis.” Science. 225, p1258-1265, 1984.[3] Purves, D, and Lichtman, J. W, “Elimination of synapses in the developing nervous
system.”, Science, 210, p153-157, 1980.[4] McKintosh F C, Kas J and Janmey P A 1995 Phys. Rev. Lett. 75 4425[5] See:
http://scienceblogs.com/purepedantry/2006/07/background_to_the_20_year_coma.php
[6] See: http://www.med.osaka-u.ac.jp/pub/molonc/www/Esignal.html[7] Stephen D. Sentura “ Microsystem Design”,2001
Acknowledgement: NSF Nanoscale Exploratory Research Award (NER), ECS-0609413, the Welch Foundation and the Strategic Partnership for Research in Nanotechnology (SPRING), and NSF NNIN Facilities at UT Austin and Stanford Center for Integrated Systems (Grant # 0335765 and 9731293 respectively)
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