An automated optical tweezers system was designed and constructed for biomolecular investigations. Key aspects included automation and control of the tweezers, calibration of stiffness and sensitivity, and DNA sample preparation and experiments. Results showed DNA overstretching and unzipping experiments in both water and heavy water. Future work will focus on further automation and investigating DNA-protein interactions.

1. An automated and user-friendly optical
tweezers for biomolecular
investigations.
By
Pranav Rathi

2. Acknowledgments
Dr. Larry Herskowitz Dr. Andy Maloney
Dr. Anthony Salvagno
Dr. Steven Koch
Collaborations
Susan Atlas—Lead of the DTRA project
UNM Physics / Cancer Center / Director of CARC
Haiqing Liu (G. Mantano lab)—Microdevice applications of kinesin
LANL & Center for Integrated Nanotechnology (CINT)
Funding DTRA—DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008

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

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

5. Optical Tweezers are used to apply forces over nanometer scale on the
order of piconewtons
F= ∆p
∆t
P
F = Qn
c
F= F +F s
2
g
2

6. Model to explain optical trap
λ≈d
Electric dipole model Electromagnetic field model Ray optics model

7. Design considerations
• Force ~ 65pN with .530nm (diameter) polystyrene
beads
• Stability and precision
• Fast, user-friendly and automated
• Safety

8. Optical path

9. Design

10. Laser-part

11. Microscope-part

12. Some problems with the design!
• Accessibility to optomechanical controls of Z lens,
QPD and microscope
• Temperature hike inside enclosure
• Mechanical vibration nose

13. Accessibility problem was solved by extending
optomechanical controls
Z-lens controls QPD controls
Microscope focus control

14. Temperature (C)
Temperature (C)
Temperature hike problem
Seconds (S)
Seconds (S)

15. Temperature hike problem was solved by
developing Fiber light
Microscope inlet Fiber plugin adapter
Fiber feeder

16. Mechanical vibration noise
Mechanical noise Airborne noise

17. Control and automation part-1

18. Control and automation part-2

19. Sample holder plate control

20. Z-stage control

21. Luca camera control
Video section
Live-feed section

22. Feedback main
Steps for data acquisition
Hard limit
parameters Journal of acquired data

23. Optical tweezers calibration

24. The parameters we calibrate!
Z
F = −Kx X
Trap center
Kx is the stiffness in x direction
X
Zb X is displacement of bead center
Beam waist from the trap center
Zb is the distance between beam
waist and the trap center.
Surface

25. Calibration of stiffness Kx
We use Brownian noise to map the stiffness
Equation of motion for trapped bead
Power spectrum
m(t ) = − β x(t ) − K x x(t ) + f (t )
x 
2
m(t ) = 0; β = 6πηr ; f (ω ) = 4 β k BT
x
After Fourier transformation
~ (ω) 2 ( K 2 + 4π 2ω 2 β 2 ) = 4 βK T
x x B
~ (ω) 2 = K BT
Cutoff frequency fc x
 K 
π 2 β ( x ) + ω 2 
 2πβ 
Kx
fc =
2πβ
6πη r
β= 3 4 5
9  r  1  r  45  r  1  r 
1−  +   −   −  
16  h  8  h  256  h  16  h 

26. 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

27. Corner frequency vs bead center height from surface (H2O)
Corner frequency (Hz)
Corner frequency (Hz)
Bead center height (multiples of r=520 nm from surface)
Bead center height (multiples of r=520 nm from surface)

28. Corner frequency vs bead center height from surface
H2O vs D2O
Corner frequency (Hz)
Corner frequency (Hz)
Bead center height (multiples of r=265 nm from surface)
Bead center height (multiples of r=265 nm from surface)

29. Stiffness vs bead center height from surface
H2O vs D2O
Perceived stiffness (pN/nm/W)
Perceived stiffness (pN/nm/W)
Bead center height (multiples of r=520 nm from surface)
Bead center height (multiples of r=520 nm from surface)
Stiffness does not depend on height but corner frequency does

30. Stiffness calibration results
Big beads (1.04µm; diameter)
Estimated stiffness (H2O) .038(7) pN/nm
Average variance (H2O) 12300+/-800 mV2
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

31. Calibration of detector sensitivity (DOG)

32. 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

33. X (mV)
X (mV) DOG at different bead heights (big beads)
Piezo (nm)
Piezo (nm)

34. X sensitivity vs bead position relative to beam waist
X Sensitivity (mV/nm)
X Sensitivity (mV/nm)
Big beads
Bead position relative to beam waist (nm)
Bead position relative to beam waist (nm)

35. Sensitivity calibration results
Sensitivity for big beads at trap center 10.8+/-.5 mV/nm
Sensitivity for small beads at trap center 2.4+/-.2mV/nm
Comparison
Sensitivity of small bead is 4.5 times the big bead and
the stiffness of big bead is 4.3 times the small bead

36. X (mV)
X (mV) Trap center verification (big beads)
Piezo (nm)
Piezo (nm)

37. X (mV)
X (mV) Trap center verification (small beads)
Piezo (nm)
Piezo (nm)

38. DNA Sample preparation

39. DNA overstretching
~65 pN

40. Results
DNA Overstretching in H2O
Force (pN)
Force (pN)
LDNA (nm)
LDNA (nm)

41. DNA Overstretching in D2O
Force (pN)
Force (pN)
LDNA (nm)
LDNA (nm)

42. Force (pN)
Force (pN)
Force D2O vs H2O
DNA tethers
DNA tethers

43. Force (pN)
DNA unzipping
Force (pN)
Piezo (nm)
Piezo (nm)

44. 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

45. Thank You

46. Appendix: A

48. Major components of laser-part
1064nm 2W
Slow shutter
AOM
Fast shutter

49. Major components of microscope-part
Microscope

50. Luca Camera
X
Z
Y
460A-XY stage
Base stage 1 & 2

51. Trap steering optics and dichroic holder stage assembly
Dichroic holder stage
Trap steering lens

52. Dichroic holder stage assembly
Mirror holder
Lens tube
holder
L4 Top view
Holder stage
Clip
Dichroic holder stage
X Z platform
Z
Y
Before reflection X Y
After reflection

53. Different parts of dichroic holder stage assembly
Slot
Clamp
Clip
Slot

54. Sample holder stage assembly
Sample holder plate
X-piezo stage
Adapter plate
X-Y translation stage
6x4 inches bread board
Sample holder stage platform

55. Sample holder plate
Rubber cushion
Lower side
Sample side
Upper side

56. Condenser and objective
Condenser
Objective
Z-Piezo
SM1L10 lens tube

57. Condenser and objective
Cube holder plate White light
LB6C plate
QPD
LCP02
adapter
SM2D25D
aperture
Dichroic holder cube LC6W
LB4C mirror holder

58. QPD sensor assembly
X
Z
Optmechanical controls
Y
Clamp QPD
QPD lens L5 STIXY translation stage
QPD platform
Base stage

59. QPD sensor assembly
LCP02 Y
X
Z
Y
QPD
LF X
ND
L5
Optmechanical controls
STIXY translation stage
QPD platform

60. Filter assembly

61. Laser-part enclosure
Service door

62. Laser-part enclosure
Service door1
Beam pipe
Service door2
Service door3

63. Optical tweezers