An automated and user-friendly optical tweezers for biomolecular

investigations.By

Pranav Rathi

Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARC

Haiqing Liu (G. Mantano lab)—Microdevice applications of kinesinLANL & Center for Integrated Nanotechnology (CINT)

Collaborations

Funding DTRA—DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008

Dr. Larry Herskowitz

Dr. Anthony Salvagno

Dr. Andy Maloney

Dr. Steven Koch

Acknowledgments

Outline

• Introduction to optical tweezers• Design and construction• Automation and control• Optical tweezers calibration• DNA sample preparation• Results

Introduction to Optical Tweezers(3-D model in Solidworks)

c

PQnF =

tpF ∆

∆=

22gs FFF +=

Optical Tweezers are used to apply forces over nanometer scale on the order of piconewtons

Model to explain optical trap

Ray optics modelElectric dipole model Electromagnetic field model

λ≈d

Design considerations

• Force ~ 65pN with .530nm (diameter) polystyrene beads

• Stability and precision

• Fast, user-friendly and automated

• Safety

Optical path

Design

Laser-part

Microscope-part

Some problems with the design!

• Accessibility to optomechanical controls of Z lens, QPD and microscope

• Temperature hike inside enclosure

• Mechanical vibration nose

Accessibility problem was solved by extending optomechanical controls

Z-lens controls QPD controls

Microscope focus control

Temperature hike problemT

empe

ratu

re (

C)

Tem

pera

ture

(C

)

Seconds (S)Seconds (S)

Fiber plugin adapterMicroscope inlet

Fiber feeder

Temperature hike problem was solved bydeveloping Fiber light

Mechanical noise Airborne noise

Mechanical vibration noise

Control and automation part-1

Control and automation part-2

Sample holder plate control

Z-stage control

Video section

Live-feed section

Luca camera control

Steps for data acquisition

Journal of acquired dataHard limitparameters

Feedback main

Optical tweezers calibration

The parameters we calibrate!

XKF x−=

Kx is the stiffness in x direction

X is displacement of bead center from the trap center

Zb is the distance between beamwaist and the trap center.

X

Z

Trap center

Beam waist

Surface

Zb

Calibration of stiffness Kx We use Brownian noise to map the stiffness

Tkfrtxm

tftxKtxtxm

B

x

βωπηβ

β

4)(;6;0)(

)()()()(2 ===

+−−=

After Fourier transformation

πβ

ωπβ

βπω

ββωπω

2

)2

(

)(~

4)4()(~

22

2

22222

xc

x

B

Bx

Kf

K

TKx

TKKx

=

+

=

=+

Power spectrumEquation of motion for trapped bead

Cutoff frequency fc

543

161

25645

81

169

1

6

−

−

+

−

=

hr

hr

hr

hr

rπηβ

Trap center determination

At 1.2r (bead radius) from surface fc≈1/2 of bulk

• Trap center offset for big beads is 186 and small bead is 367 nm• Big bead is 1.96 times the small bead and small bead is 1.97 times farther then big bead

Corner frequency vs bead center height from surface (H2O)C

orne

r fr

eque

ncy

(Hz)

Cor

ner

freq

uenc

y (H

z)

Bead center height (multiples of r=520 nm from surface)Bead center height (multiples of r=520 nm from surface)

Corner frequency vs bead center height from surfaceH2O vs D2O

Cor

ner

freq

uenc

y (H

z)C

orne

r fr

eque

ncy

(Hz)

Bead center height (multiples of r=265 nm from surface)Bead center height (multiples of r=265 nm from surface)

Stiffness vs bead center height from surfaceH2O vs D2O

Stiffness does not depend on height but corner frequency does

Perc

eive

d st

iffn

ess

(pN

/nm

/W)

Perc

eive

d st

iffn

ess

(pN

/nm

/W)

Bead center height (multiples of r=520 nm from surface)Bead center height (multiples of r=520 nm from surface)

Estimated stiffness (H2O) .038(7) pN/nm

Average variance (H2O) 12300+/-800 mV2

Stiffness calibration results

Big beads (1.04µm; diameter)

Estimated stiffness (D2O) .04(2) pN/nm

Average variance (D2O) 12500+/800 mV2

Small beads (.530µm; diameter)

Estimated stiffness (H2O) .011(5) pN/nm

Average variance (H2O) 2100+/-200 mV2

Estimated stiffness (D2O) .012(5) pN/nm

Average variance (D2O) 2000+/-300 mV2

Calibration of detector sensitivity (DOG)

Calibration of detector sensitivity (DOG)

DOG scan and linear-fit on left and sensitivity extracted from slope of linear-fit vs bead position relative to beam waist on right from

DOG scans from one edge to another of bead

DOG at different bead heights (big beads)X

(m

V)

X (

mV

)

Piezo (nm)Piezo (nm)

X sensitivity vs bead position relative to beam waist

Big beads

X S

ensi

tivi

ty (

mV

/nm

)X

Sen

siti

vity

(m

V/n

m)

Bead position relative to beam waist (nm)Bead position relative to beam waist (nm)

Sensitivity calibration results

Sensitivity of small bead is 4.5 times the big bead and the stiffness of big bead is 4.3 times the small bead

Comparison

Sensitivity for big beads at trap center 10.8+/-.5 mV/nm

Sensitivity for small beads at trap center 2.4+/-.2mV/nm

Trap center verification (big beads)X

(m

V)

X (

mV

)

Piezo (nm)Piezo (nm)

Trap center verification (small beads)X

(m

V)

X (

mV

)

Piezo (nm)Piezo (nm)

DNA Sample preparation

DNA overstretching

~65 pN

ResultsDNA Overstretching in H2O

Forc

e (p

N)

For

ce (

pN)

LDNA (nm)LDNA (nm)

DNA Overstretching in D2OF

orce

(pN

)Fo

rce

(pN

)

LDNA (nm)LDNA (nm)

Force D2O vs H2OF

orce

(pN

)F

orce

(pN

)

DNA tethersDNA tethers

DNA unzippingF

orce

(pN

)F

orce

(pN

)

Piezo (nm)Piezo (nm)

Future work

• Automate Z piezo using camera to find the surface

• Automate Z lens and QPD controls

• DNA unzipping in D2O

• Investigate DNA protein interactions in H2O and D2O

• Develop touch screen controlled automation for optical tweezers

Thank You

Appendix: A

Major components of laser-part

1064nm 2W

Slow shutter

AOM

Fast shutter

Major components of microscope-partMicroscope

Luca Camera

Base stage 1 & 2

460A-XY stage

X

Y

Z

Trap steering optics and dichroic holder stage assembly

Trap steering lens

Dichroic holder stage

Holder stageL4

Mirror holderLens tube holder

Dichroic holder stage platform

Top view

Clip

X

Z

YAfter reflection

X Z

YBefore reflection

Dichroic holder stage assembly

Slot

Slot

Clip

Clamp

Different parts of dichroic holder stage assembly

Sample holder plate

X-piezo stage

X-Y translation stage

Sample holder stage platform

Adapter plate

6x4 inches bread board

Sample holder stage assembly

Sample holder plate

Upper side

Lower side

Sample side

Rubber cushion

Objective

Condenser

Z-PiezoSM1L10 lens tube

Condenser and objective

Cube holder plate White light

QPD

Dichroic holder cube LC6W

LB6C plate

SM2D25Daperture

LCP02adapter

LB4C mirror holder

Condenser and objective

XZ

Y

QPD lens L5 STIXY translation stageQPD

QPD platform

Optmechanical controls

Base stage

Clamp

QPD sensor assembly

XZ

YQPD

Y

X

L5

STIXY translation stage

Optmechanical controls

LCP02

QPD platform

LFND

QPD sensor assembly

Filter assembly

Service door

Laser-part enclosure

Service door1

Service door2

Service door3

Beam pipe

Laser-part enclosure

Optical tweezers