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An automated and user-friendly optical tweezers for biomolecular investigations (PhD Defense)
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.
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An automated and user-friendly optical tweezers for biomolecular investigations (PhD Defense)
- 1. An automated anduser-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 tooptical tweezers • Design and construction • Automation and control • Optical tweezers calibration • DNA sample preparation • Results
- 4. Introduction to OpticalTweezers (3-D model in Solidworks)
- 5. Optical Tweezers areused 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 explainoptical 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
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- 12. Some problems withthe design! • Accessibility to optomechanical controls of Z lens, QPD and microscope • Temperature hike inside enclosure • Mechanical vibration nose
- 13. Accessibility problem wassolved 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 problemwas solved by developing Fiber light Microscope inlet Fiber plugin adapter Fiber feeder
- 16. Mechanical vibration noise Mechanicalnoise Airborne noise
- 17. Control and automationpart-1
- 18. Control and automationpart-2
- 19. Sample holder platecontrol
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- 21. Luca camera control Videosection Live-feed section
- 22. Feedback main Steps for data acquisition Hard limit parameters Journal of acquired data
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- 24. The parameters wecalibrate! 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 stiffnessKx 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 vsbead 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 vsbead 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 beadcenter 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 detectorsensitivity (DOG)
- 32. Calibration of detectorsensitivity (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 vsbead 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 Sensitivityfor 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)
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- 39. DNA overstretching ~65 pN
- 40. Results DNA Overstretching in H2O Force (pN) Force (pN) LDNA (nm) LDNA (nm)
- 41. DNA Overstretching inD2O 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 • AutomateZ 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
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- 48. Major components oflaser-part 1064nm 2W Slow shutter AOM Fast shutter
- 49. Major components ofmicroscope-part Microscope
- 50. Luca Camera X Z Y 460A-XY stage Base stage 1 & 2
- 51. Trap steering opticsand dichroic holder stage assembly Dichroic holder stage Trap steering lens
- 52. Dichroic holder stageassembly 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 ofdichroic holder stage assembly Slot Clamp Clip Slot
- 54. Sample holder stageassembly Sample holder plate X-piezo stage Adapter plate X-Y translation stage 6x4 inches bread board Sample holder stage platform
- 55. Sample holder plate Rubbercushion 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
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- 61. Laser-part enclosure Service door
- 62. Laser-part enclosure Service door1 Beam pipe Service door2 Service door3
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