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An automated and user-friendly optical      tweezers for biomolecular           investigations.                  By       ...
Acknowledgments               Dr. Larry Herskowitz                         Dr. Andy Maloney               Dr. Anthony Salv...
Outline• Introduction to optical tweezers• Design and construction• Automation and control• Optical tweezers calibration• ...
Introduction to Optical Tweezers      (3-D model in Solidworks)
Optical Tweezers are used to apply forces over nanometer scale on the                        order of piconewtons         ...
Model to explain optical trap                                 λ≈dElectric dipole model   Electromagnetic field model   Ray...
Design considerations• Force ~ 65pN with .530nm (diameter) polystyrenebeads• Stability and precision• Fast, user-friendly ...
Optical path
Design
Laser-part
Microscope-part
Some problems with the design!• Accessibility to optomechanical controls of Z lens, QPD and microscope• Temperature hike i...
Accessibility problem was solved by extending           optomechanical controls        Z-lens controls                    ...
Temperature (C)Temperature (C)                  Temperature hike problem                            Seconds (S)           ...
Temperature hike problem was solved by        developing Fiber light        Microscope inlet    Fiber plugin adapter      ...
Mechanical vibration noiseMechanical noise               Airborne noise
Control and automation part-1
Control and automation part-2
Sample holder plate control
Z-stage control
Luca camera controlVideo section  Live-feed section
Feedback main             Steps for data acquisitionHard limitparameters                       Journal of acquired data
Optical tweezers calibration
The parameters we calibrate!    Z                                        F = −Kx X         Trap center                    ...
Calibration of stiffness Kx           We use Brownian noise to map the stiffness                                         E...
Trap center determination                  At 1.2r (bead radius) from surface fc≈1/2 of bulk• Trap center offset for big b...
Corner frequency vs bead center height from surface (H2O)Corner frequency (Hz)Corner frequency (Hz)                       ...
Corner frequency vs bead center height from surface                                           H2O vs D2OCorner frequency (...
Stiffness vs bead center height from surface                                                 H2O vs D2OPerceived stiffness...
Stiffness calibration results         Big beads (1.04µm; diameter)Estimated stiffness (H2O)   .038(7) pN/nmAverage varianc...
Calibration of detector sensitivity (DOG)
Calibration of detector sensitivity (DOG)DOG scan and linear-fit on left and sensitivity extracted from slope of  linear-f...
X (mV)X (mV)   DOG at different bead heights (big beads)                            Piezo (nm)                            ...
X sensitivity vs bead position relative to beam waistX Sensitivity (mV/nm)X Sensitivity (mV/nm)                           ...
Sensitivity calibration resultsSensitivity for big beads at trap center   10.8+/-.5 mV/nmSensitivity for small beads at tr...
X (mV)X (mV)   Trap center verification (big beads)                        Piezo (nm)                         Piezo (nm)
X (mV)X (mV)   Trap center verification (small beads)                          Piezo (nm)                           Piezo ...
DNA Sample preparation
DNA overstretching                     ~65 pN
Results             DNA Overstretching in H2OForce (pN)Force (pN)                       LDNA (nm)                        L...
DNA Overstretching in D2OForce (pN)Force (pN)                       LDNA (nm)                        LDNA (nm)
Force (pN)Force (pN)             Force D2O vs H2O                   DNA tethers                   DNA tethers
Force (pN)             DNA unzippingForce (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• In...
Thank You
Appendix: A
Major components of laser-part                                 1064nm 2W                                 Slow shutter     ...
Major components of microscope-part             Microscope
Luca Camera                       X                           Z                           Y460A-XY stage    Base stage 1 & 2
Trap steering optics and dichroic holder stage assembly                                             Dichroic holder stageT...
Dichroic holder stage assembly         Mirror holderLens tubeholder          L4                                           ...
Different parts of dichroic holder stage assembly                                       Slot                  Clamp       ...
Sample holder stage assembly                      Sample holder plate              X-piezo stage              Adapter plat...
Sample holder plateRubber cushion                 Lower side                              Sample side                 Uppe...
Condenser and objective                  Condenser                   ObjectiveZ-Piezo                 SM1L10 lens tube
Condenser and objective                 Cube holder plate             White lightLB6C plate                               ...
QPD sensor assembly      X          Z                                                 Optmechanical controls     Y    Clam...
QPD sensor assemblyLCP02                  Y                           X                               Z                   ...
Filter assembly
Laser-part enclosure         Service door
Laser-part enclosure                            Service door1Beam pipe                                  Service door2     ...
Optical tweezers
An automated and user-friendly optical  tweezers for biomolecular  investigations (PhD Defense)
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An automated and user-friendly optical tweezers for biomolecular investigations (PhD Defense)

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An automated and user-friendly optical tweezers for biomolecular investigations; a versatile, automated, fast, precise and user friendly optical tweezers capable of doing verity of biomolecular experiments.

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An automated and user-friendly optical tweezers for biomolecular investigations (PhD Defense)

  1. 1. An automated and user-friendly optical tweezers for biomolecular investigations. By Pranav Rathi
  2. 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. 3. Outline• Introduction to optical tweezers• Design and construction• Automation and control• Optical tweezers calibration• DNA sample preparation• Results
  4. 4. Introduction to Optical Tweezers (3-D model in Solidworks)
  5. 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. 6. Model to explain optical trap λ≈dElectric dipole model Electromagnetic field model Ray optics model
  7. 7. Design considerations• Force ~ 65pN with .530nm (diameter) polystyrenebeads• Stability and precision• Fast, user-friendly and automated• Safety
  8. 8. Optical path
  9. 9. Design
  10. 10. Laser-part
  11. 11. Microscope-part
  12. 12. Some problems with the design!• Accessibility to optomechanical controls of Z lens, QPD and microscope• Temperature hike inside enclosure• Mechanical vibration nose
  13. 13. Accessibility problem was solved by extending optomechanical controls Z-lens controls QPD controls Microscope focus control
  14. 14. Temperature (C)Temperature (C) Temperature hike problem Seconds (S) Seconds (S)
  15. 15. Temperature hike problem was solved by developing Fiber light Microscope inlet Fiber plugin adapter Fiber feeder
  16. 16. Mechanical vibration noiseMechanical noise Airborne noise
  17. 17. Control and automation part-1
  18. 18. Control and automation part-2
  19. 19. Sample holder plate control
  20. 20. Z-stage control
  21. 21. Luca camera controlVideo section Live-feed section
  22. 22. Feedback main Steps for data acquisitionHard limitparameters Journal of acquired data
  23. 23. Optical tweezers calibration
  24. 24. The parameters we calibrate! Z F = −Kx X Trap center Kx is the stiffness in x directionX 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. 25. Calibration of stiffness Kx We use Brownian noise to map the stiffness Equation of motion for trapped beadPower 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. 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. 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. 28. Corner frequency vs bead center height from surface H2O vs D2OCorner 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. 29. Stiffness vs bead center height from surface H2O vs D2OPerceived 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. 30. Stiffness calibration results Big beads (1.04µm; diameter)Estimated stiffness (H2O) .038(7) pN/nmAverage variance (H2O) 12300+/-800 mV2Estimated stiffness (D2O) .04(2) pN/nmAverage variance (D2O) 12500+/800 mV2 Small beads (.530µm; diameter)Estimated stiffness (H2O) .011(5) pN/nmAverage variance (H2O) 2100+/-200 mV2Estimated stiffness (D2O) .012(5) pN/nmAverage variance (D2O) 2000+/-300 mV2
  31. 31. Calibration of detector sensitivity (DOG)
  32. 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. 33. X (mV)X (mV) DOG at different bead heights (big beads) Piezo (nm) Piezo (nm)
  34. 34. X sensitivity vs bead position relative to beam waistX Sensitivity (mV/nm)X Sensitivity (mV/nm) Big beads Bead position relative to beam waist (nm) Bead position relative to beam waist (nm)
  35. 35. Sensitivity calibration resultsSensitivity for big beads at trap center 10.8+/-.5 mV/nmSensitivity 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. 36. X (mV)X (mV) Trap center verification (big beads) Piezo (nm) Piezo (nm)
  37. 37. X (mV)X (mV) Trap center verification (small beads) Piezo (nm) Piezo (nm)
  38. 38. DNA Sample preparation
  39. 39. DNA overstretching ~65 pN
  40. 40. Results DNA Overstretching in H2OForce (pN)Force (pN) LDNA (nm) LDNA (nm)
  41. 41. DNA Overstretching in D2OForce (pN)Force (pN) LDNA (nm) LDNA (nm)
  42. 42. Force (pN)Force (pN) Force D2O vs H2O DNA tethers DNA tethers
  43. 43. Force (pN) DNA unzippingForce (pN) Piezo (nm) Piezo (nm)
  44. 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 foroptical tweezers
  45. 45. Thank You
  46. 46. Appendix: A
  47. 47. Major components of laser-part 1064nm 2W Slow shutter AOM Fast shutter
  48. 48. Major components of microscope-part Microscope
  49. 49. Luca Camera X Z Y460A-XY stage Base stage 1 & 2
  50. 50. Trap steering optics and dichroic holder stage assembly Dichroic holder stageTrap steering lens
  51. 51. Dichroic holder stage assembly Mirror holderLens tubeholder L4 Top view Holder stage Clip Dichroic holder stage X Z platform Z YBefore reflection X Y After reflection
  52. 52. Different parts of dichroic holder stage assembly Slot Clamp Clip Slot
  53. 53. Sample holder stage assembly Sample holder plate X-piezo stage Adapter plate X-Y translation stage 6x4 inches bread board Sample holder stage platform
  54. 54. Sample holder plateRubber cushion Lower side Sample side Upper side
  55. 55. Condenser and objective Condenser ObjectiveZ-Piezo SM1L10 lens tube
  56. 56. Condenser and objective Cube holder plate White lightLB6C plate QPD LCP02 adapterSM2D25Daperture Dichroic holder cube LC6W LB4C mirror holder
  57. 57. QPD sensor assembly X Z Optmechanical controls Y Clamp QPD QPD lens L5 STIXY translation stage QPD platformBase stage
  58. 58. QPD sensor assemblyLCP02 Y X Z Y QPD LF X ND L5 Optmechanical controls STIXY translation stage QPD platform
  59. 59. Filter assembly
  60. 60. Laser-part enclosure Service door
  61. 61. Laser-part enclosure Service door1Beam pipe Service door2 Service door3
  62. 62. Optical tweezers

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