use of tethering for axial confinement in optical tweezers mark cronin-golomb biomedical engineering...
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Use of tethering for axial confinement in optical tweezers
Mark Cronin-Golomb
Biomedical Engineering
Tufts University
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Outline
Motivation Design of DNA tether Videos of untethered and tethered particles Confocal detection measurement system Demonstration of force measurement Future directions
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Tethers and tweezers
Microspheres tethered to each other (Chu)
Backscattering from tethered bead as probe of DNA flexibility (Libchaber APL 73, 291 (1998))
Twisting polymers by applying torque to trapped particle (Bustamante Nature 424, 338
(2003), Ormos) Study of macromolecular motion (Gelles)
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Use of low numerical aperture trapping lenses Trapping particles
against glass slide
Trapping against counterflow
Trapping against gravity
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Axial trapping is harder to achieve than transverse trapping Generalized Lorenz-Mie theory to find
radiation pressure cross section Cpr(z) and radiation pressure force F in terms of standard Mie scattering coefficients:
220
2( )pr
n PF C z
c w
K.F. Ren, G. Gréhan, and G. Gouesbet, Appl. Opt. 35, 2702 (1996)
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Axial force with 1.25NA beam
-30
-20
-10
0
10
20
30
-1.0 -0.5 0.0 0.5 1.0
Axial Position (m)
Axi
al F
orc
e (p
N)
1m diameter polystyrene bead, 13mW 820nm wavelength trap
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Axial force for 0.65NA beam
-20
-15
-10
-5
0
5
10
15
20
-10 -5 0 5 10
Axial Position (m)
Axi
al F
orc
e (p
N)
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Beads in 0.65NA trap without tether
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Comparison of original and tethered configurations
NA1.3
Trap Beam
Bead
Trap Beam
Bead
DNA48k base pairs
31.5x106 Dalton
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Experiment Details
Ti - Sapphire Laser
Lock - in Amplifier
CCD Camera
OL
DM
P APD
*
*
AOD Driver
BS
*
AOD
#
#
#
SM
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No dCTPstop
No dCTPstop
1. dNTPs – dCTP + biotin-dUTP + Klenow
2. + dCTP + digoxigenin-dUTP
C|C|
G|C|
G|G|
AGGTTACG
TCCAATGCCCCGCCGCTGGA||||||||
G|G|G|C|
A|
U|
DIG|
C|C|C|G|C|C|G|
G|
GGGCGGCGACCTCGCGGGTT
GCGCCCAA||||||||A|
G|
U|
biotin|C|
Zimmermann and Cox, Nucleic Acids Research 22, 492 (1994)
End labeling DNA for attachment to streptavidin and anti-digoxigenin
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Tether construct
Streptavidin
Biotin
DIG
Goat anti-mouse IgG bead
Mouse anti-DIG antibody
Cover slip
Modified from Meiners and QuakePhys. Rev. Lett. 84, 5014 (2000)
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Frame sequence from tethered bead video
10 m
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Tethered beads in 0.65NA trap
Tracking Software implemented in IDL by Crocker and Weeks
http://www.physics.emory.edu/~weeks/idl/
10m
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Experiment Details: measurements
Ti - Sapphire Laser
Lock - in Amplifier
CCD Camera
OL
DM
P APD
*
*
AOD Driver
BS
*
AOD
#
#
#
SM
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•As the tweezer beam is moved back and forth, the probe bead lags behind.
•The bead is bright when the tweezer beam illuminates it.
•The confocal signal is highest when the tweezer beam is centered on the probe bead.
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At large oscillation amplitudes the potential well splits
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Theoretical Background
sin( ) ( )dx
x a t L tdt
x: trap position : viscous drag
: tweezer spring constant a: amplitude of trap oscillation
: frequency of trap oscillation L(t): Brownian forcing function
2
cos
sin
( ) 1 ( )
duu a t
dtu x a t
I t u t
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0 1000 2000 3000 4000 0
5
10
15
20
25
30
SNR
~ f -3/2
~ f -1/2 Confocal
Split PD
frequency (Hz)
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Viscosity Image
Viscosity distribution around A. pullulans imaged by raster scanning an optically trapped probe bead.
This blastospore has a halo of the polysaccharide pullulan around it. Note the viscosity gradient.
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Force Off
Oscillating Laser Trap
Probe Bead Probe Bead
a
r
Force On
Probe BeadProbe Bead
aOscillating Laser Trap
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We can use confocal tweezers to measure forces applied to probe beads.
Flow measurement is one example of force measurement
Force Measurement
1
1/ 222
16
41
R aF r
R
2cot / 2
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Force measurement
An optically trapped microsphere is used as a probe for two-dimensional force imaging using scanning optics.
A fluid viscosity map may be obtained simultaneously. Calibration is based on a single length measurement only:
the oscillation amplitude a of the trap.
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Transverse force on tethered bead
0.01
0.10
1.00
10.00
100.00
5 10 15 20 25 30
Position (um)
Fo
rce
(pN
)
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Further applications
Fiber based sensor
Laser in
Detector and electronics
Piezotransducer
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Applications
Photonic force microscope with retained probe bead
Measurement of changes in tether properties with environment, e.g. with enzymes, buffer properties etc.
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Array of tethered beads for actin network network generation and analysis
From Christian Schmitz’ talk
Actin
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Conclusions
Probe beads can be tethered to substrates to eliminate need for axial trapping, enabling use of low NA objectives.
Measurements of viscosity and force can be made with tethered beads via confocal detection system
References to confocal detection method: Nemet, Shabtai, Cronin-Golomb, Opt. Lett. 27, 264 (2002) Nemet, Cronin-Golomb, Opt. Lett. 27, 1357, (2002) Nemet, Cronin-Golomb, Appl. Opt. 42, 1820 (2003)
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Acknowledgements
Boaz Nemet Joe Platko Support of Tufts University Bioengineering
Center