Use of tethering for axial confinement in optical tweezers

Mark Cronin-Golomb

Biomedical Engineering

Tufts University

Outline

Motivation Design of DNA tether Videos of untethered and tethered particles Confocal detection measurement system Demonstration of force measurement Future directions

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)

Use of low numerical aperture trapping lenses Trapping particles

against glass slide

Trapping against counterflow

Trapping against gravity

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)

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

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)

Beads in 0.65NA trap without tether

Comparison of original and tethered configurations

NA1.3

Trap Beam

Bead

Trap Beam

Bead

DNA48k base pairs

31.5x106 Dalton

Experiment Details

Ti - Sapphire Laser

Lock - in Amplifier

CCD Camera

OL

DM

P APD

*

*

AOD Driver

BS

*

AOD

#

#

#

SM

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

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)

Frame sequence from tethered bead video

10 m

Tethered beads in 0.65NA trap

Tracking Software implemented in IDL by Crocker and Weeks

http://www.physics.emory.edu/~weeks/idl/

10m

Experiment Details: measurements

Ti - Sapphire Laser

Lock - in Amplifier

CCD Camera

OL

DM

P APD

*

*

AOD Driver

BS

*

AOD

#

#

#

SM

•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.

At large oscillation amplitudes the potential well splits

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

0 1000 2000 3000 4000 0

5

10

15

20

25

30

SNR

~ f -3/2

~ f -1/2 Confocal

Split PD

frequency (Hz)

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.

Force Off

Oscillating Laser Trap

Probe Bead Probe Bead

a

r

Force On

Probe BeadProbe Bead

aOscillating Laser Trap

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

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.

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

)

Further applications

Fiber based sensor

Laser in

Detector and electronics

Piezotransducer

Applications

Photonic force microscope with retained probe bead

Measurement of changes in tether properties with environment, e.g. with enzymes, buffer properties etc.

Array of tethered beads for actin network network generation and analysis

From Christian Schmitz’ talk

Actin

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)

Acknowledgements

Boaz Nemet Joe Platko Support of Tufts University Bioengineering

Center