Design and Documentation (OTZ)

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Contents

Overview of Optical Trapping Lab

The idea of using light beams as tweezers to manipulate cells and tiny objects has been applied widely in physics and biology, including measuring forces generated by molecular motors and forces required to unzip DNA double helices. Optical forces generated by a laser beam focused through a high numerical aperture lens can apply pico-Newton forces and provide nanometer position resolution, providing a powerful tool for moving, controlling and measuring objects. In this lab, students learn the fundamentals of optical trapping, learn to calibrate an optical trap for position detection and force measurement using synthetic beads, and apply the technique to study internal transport by molecular motors in onion cells and flagellar locomotion in E. Coli cells. The lab writeup is currently in a preliminary draft form at Optical Trapping.

Acknowledgements

This experiment was developed under the direction of Professor Jan Liphardt. Suneet Upadhyay, an undergraduate Segre intern and student assistant, built, tested, and refined the optical trap patterned on David Appleyard's design at MIT. Suneet wrote the C#/.NET data acquisition and control application. The apparatus and staff time were funded by a generous gift from the Microsoft Research Division.

Apparatus

The optical trap designed by Dave Appleyard at MIT is described on his web site and published in Optical Trapping for Undergraduates. American Journal of Physics, Vol. 75, No. 1, pp 5-14, January 2007. He developed this for MIT's course 20.309, Biological Engineering II: Instrumentation and Measurement. Both MIT and Berkeley are continuing to refine this design.

Revised optical trap layout at Berkeley

Modifications at Berkeley parallel those at MIT but do not go as far. Optical components of the beam path and detector branch have been mounted in a cage system, but most of the microscope and camera branch have not. The 3D stage in the original design required a higher force than the picomotors are rated for, leading to burned out motors, so this was replaced with two 1D stages (Newport 423). For a microscope illuminator, we use a blue LED (Luxeon LXK2-PB14-N00) attached to a heat sink.

Suneet Upadyay started our optical trap in Summer 2007
Front view
Substage area
Top view

Revised optical trap layout at MIT

In an effort to make the optical trap easier to align and protect the beam path without enclosing the trap in a box, all of the optical components have been mounted in a cage system with lens tubes. The revised trap was used by Thorlabs in late 2008 as a model for their optical trap kit, now available with a single part number from Thorlabs. The MIT trap, refined by Steven Wasserman, is pictured below.

Front view
Top view


Setup and Maintenance

Laser Replacement

Precautions: ALWAYS WEAR A GROUNDING BRACELET WHEN TOUCHING THE LASER OR ANY COMPONENT IN CONTACT WITH IT. Static discharge will destroy the laser. Also make sure not to kink or break the laser optic. If the fiber optic is bent more sharply then the minimum radius of curvature it will destroy it.

Due to the delicacy of the laser, it is advised that whenever dealing with a laser the user manuals for the TEC, LDC, Laser Mount and Laser all be read to make sure the functionality of each is understood well. These instructions are not complete, but give a general outline of how to proceeed.

Currently a 200mW Lumics laser is mounted in a Thorlabs LM14 laser mount. The card in the laser mount is specific to the laser design. Most diode lasers use the same configuration, but the correct card is necessary as a wrong card will deliver voltages and currents incorrectly to the laser and likely destroy it. The TEC is a Thorlabs TEC2000 and controls the temperature of the laser diode, preventing overheating. The LDC is the Thorlabs LDC 2000 and sends power to the laser and actually drives the laser. If too much power is sent to the laser it will immediately be destroyed. The laser mount allows the outputs on the TEC and LDC to be matched up to the correct pins on the butterfly laser diode. The laser diode has 14 pins which must each be driven correctly for the laser to function properly. The LDC and TEC are on long term loan from Jan Liphardt, whereas he has given the laser to the lab.


  1. First check to see how each pin in the 9 pin outputs of the LDC and TEC are driven. The manuals for the TEC and LDC each give a diagram of how each of the 9 pin outputs are driven.
  2. Check the laser manual to see which pin of the laser requires which input. The outputs of the LDC and TEC must drive the correct laser pin.
  3. The laser mount is the final piece of the puzzle, which must be configured to deliver the correct signal from the TEC and LDC to the correct laser pin. Check the laser mount to see if the 18 input pins (9 for each the TEC and LDC ) will correctly deliver power to the laser pins as specified by the laser manual and the TEC and LDC manuals. The manual for the mount may give incorrect pin assignments as the cards have been swapped out. To insure proper functionality, check which input corresponds to which contact by measuring the resistance between the contact and the input on the laser mount alone.
  4. If all pins are correctly assigned, the laser may be inserted into the mount according to operating procedures in the laser and mount manuals. WEAR A GROUNDING BRACELET!!! Insure during assembly that the laser fiber optic is not broken or kinked. This will cause backwards reflections into the laser which could destroy it. A good idea now, is to make sure that nothing is bridging the gap between the two wires sticking up vertically from the laser mount, located behind the laser. This insures that the laser power cannot be delivered unless the TEC is on.
  5. Before attaching any components, the TEC and LDC controllers should be correctly set so as not to exceed any maximum laser power inputs. These can be set by consulting the Absolute Maximum Ratings in the laser manual and then correctly setting the TEC and LDC controllers to correspond to this. In addition, while setting these maximum ratings, the power input to the laser diode on the LDC should be set to zero, so that there is no sudden power surge delivered to the laser.
  6. Finally, once the TEC and LDC are set to driving the laser correctly, the laser is correctly mounted and its beam sent into a safe region, the TEC and LDC hooked into the laser mount, testing may begin.

Laser Alignment and Collimation

If the beam is not centered and vertical within the microscope or does not fill the aperture of the objective lens, then the laser will fail to trap particles or trap them only against the slide. When the beam is properly aligned, a trapped particle may be moved vertically and horizontally without escaping the trap.

Safety Note: Alignment of the laser requires bypassing the safety interlock on the lid of the shielding box, creating a potential exposure hazard to a 200 mW laser with an invisible beam. This work may be performed only by individuals who have completed the campus laser safety course and taken a baseline eye exam. Appropriate safety goggles must be worn during the alignment procedure.

Useful Tools

Useful alignment aids

Targets that can be mounted on 1" ports or hung on cage bars to indicate the optical axis are indispensable. The targets pictured here are all available from Thorlabs. Target numbers indicated are referred to in the instructions below. In addition, a 6" lens tube and 1" irises that fit the lens tube are handy to have. A handheld IR video viewer is used to make the beam visible.

Procedure

A. Collimate laser

  1. To collimate laser, place cage containing laser on table so that beam projects out the door and across the BSC lab to the window (do outside of lab hours, block door for safety). Attach sheet of paper to a box to serve as target for laser. With one person observing laser projected on target (through viewer), second person adjusts distance between fiber terminator and collimating lens to focus beam. Check to see that beam is collimated along full length, with no waist. With laser power at lowest level giving a visible spot, the best that could be attained in Nov. 2009 was a beam diameter expanding from 3 mm near the laser to 8 mm across the room.

B. Align laser with beam path cage

  1. Attach laser terminator/collimator cage to beam path cage. Remove beam expander.
  2. Check laser alignment to optical axis of first leg of beam path. As an aid, hang target 3 from cage between laser terminator and collimating lens. Insert beam expander and check alignment again.

C. Align beam through cube 1.

  1. Remove dichroic mirror in cube 1 (behind microscope). Hang target 3 from cage between laser terminator and collimating lens to give sharp edge to beam.
  2. Attach target 2 to left port of steering mirror 2 and adjust steering mirror 2 to bring beam to center of target. Move target 2 to left port of turret 1 and adjust steering mirror 1 to move beam AWAY from center in both axes.
  3. Repeat step previous step until beam is centered through this leg of beam path. Check to make sure there is no clipping of the beam when target 3 is removed from cage near the laser.

D. Align beam through microscope

  1. Insert dichroic mirror in cube 1; tighten nylon screws a little to allow smooth rotation of mirror. Remove detector branch cage (containing turret 2, QPD, etc) and condenser lens. Remove slide holder from stage to provide easier access and viewing. Remove objective lens and replace with adapter for Nikon objective threads to SM1 threads.
  2. If 45 degree mirror has not already been aligned, position it below objective lens holder so that beam is reflected up through center of microscope. Mirror can be moved up and down on post to move beam in Y direction (front to back). Rotation of post is needed to make beam vertical, but does not allow adjustment of beam in X direction (as beam will no longer be vertical). The mirror positioning can be combined with the alignments in the following step.
  3. Attach target 2 to objective holder and adjust turret 1 to center the beam. (Rotate turret to adjust in X direction, turn center set screw on top of turret to adjust in Y direction). Remove target 2 and replace with a 6-inch lens tube containing target 2 on top. Adjust screws of steering mirror 2 to move beam AWAY from center in X and Y directions.
  4. Repeat previous step until beam is centered through the microscope. Check to make sure there is no clipping of the beam when target 3 is removed from cage near the laser.
  5. Tighten the four nylon screws that attach turret to cube 1. To avoid rotating the turret during the tightening, tighten each screw only slightly each time, alternating screws across from each other followed by in sequence around the turret. Check the alignment again to be sure no movement occurred. Remove targets and lens tube. At this point, the critical part of alignment that determines trapping ability is completed.
Adjust turret 1 with target on objective holder.
Adjust steering mirror 2 with target on top of lens tube.

E. Align Condenser and Detector Branch

  1. To check alignment of cube 2, attach the detector cage including cube 2 with the objective, condenser, and dichroic mirror still out. Insert target 1 into objective holder (with adapter for threads). Attach an iris to each end of the 6-inch lens tube and screw tube into top port of cube two. With room light only (laser OFF), sight down through the two irises to see if cube 2 is aligned with target 2. Adjust cage attachment to move left and right; rotate whole assembly on the large post to move front to back.
  2. Insert objective and condenser (which requires removing detector branch cage). Dichroic mirror is still removed from cube 2.
  3. Focusing condenser: Ideally the condenser height would be adjusted so that the laser beam is collimated all the way to the ceiling of the room. However, it is not possible with this condenser. One could focus beam to converge on a small spot on the ceiling, but condenser is then within 1 mm of the slide, which is too close to allow easy access for slide loading. The important thing is for the beam to be somewhat focused on the QPD, accomplished with a +35mm lens in the detector branch. The condenser height can be set near the top of the range to allow easier manipulation of the slide on the stage. Steve Wasserman recommends a geometry that images the back focal plane onto the QPD using the thin lens equation. Using the condenser diaphragm as the object, \frac{1}{S_1} + \frac{1}{S_2}= \frac{1}{f} where S1 = a + b in the diagram.
  4. Centering condenser: Attach target 2 to the top port of cube 2. Close down condenser diaphragm until circle of laser beam is visible within target. Center condenser with X-Y centering screws on condenser mount.
  5. Align beam to QPD. Remove target 2 from cube 2 and insert dichroic mirror turret. Remove QPD and in its place mount target 2 in a cage plate so target is approximately at same position as the QPD. Turn turret 2 to center beam vertically on target and tighten screws. Beam should be close to centered horizontally.
  6. Replace QPD. Fine adjustment of beam on QPD is routinely done during each experiment by adjustment of X-Z screws on cage plate holding QPD to zero the voltage recorded from the QPD.

Old Alignment Procedure

  1. Remove the beam expander and any other lenses except the collimation lens mounted adjacent to the laser fiber. Remove the the mirror that directs beam up through microscope (vertical mirror) and the objective lens, and turn the LED/QPD arm aside by loosening the large screw around the post. There is a collar below the vertical mirror and the arm to maintain the height. Do not remove either collar.
  2. Turn on the laser and point the optical fiber/collimation lens at a point as far away as you can manage (at MIT they do about 30 or 40 feet). You may find that with one person, it is only feasible to have the laser shooting out 10 or 15 feet, after which the point is out of focus. Adjust the collimation lens to get the smallest, sharpest spot possible. The laser should now be collimated. (TMake sure you are wearing laser safety goggles and that no one else is in the lab. Block all entrances to the beam path and make sure it does not leave the room!)
  3. Center beam in the cage that normally contains the beam expander. To do this, use two irises mounted in this cage and adjust the two steering mirrors.
  4. Put in the beam expander and bring the beam back to the center again with the steering mirrors.
  5. Insert the dichroic mirror and the vertical mirror . Screw the calibration tube with two irises into the objective lens holder. Adjust the dichroic mirror and vertical mirror to get beam centered in calibration tube with irises closed down. Beam should be 6mm wide to fill objective.
  6. Remove calibration tube and screw in objective lens. swing condenser into optical path. Project laser on ceiling and focus sharply by moving condenser up and down.
  7. Put a slide of large beads on the stage. Use the camera to view the laser (may need to trap bead to see it) and adjust mirror by camera to get field of view centered on the trap.
  8. Trap a bead and adjust focus of beam expander so that bead is just below the optical focus.
  9. Adjust QPD to center beam on QPD.

Software

We are currently testing two ways of controlling and taking data from the optical trap. The first is a series of LabVIEW virtual instruments developed at MIT. The second is a program using a framework and modules in C# and .NET.

LabVIEW Programs

These test and calibration programs are avilable in a single package: Zip file.

C#/.NET Program

The program is available for download as OpticalTrap1.0 Zip file.

Flowchart for software

In this series of experiments, students use an optical trap to calibrate an optical trap for position detection and force measurement using synthetic beads and apply the technique to study bacterial motility. The software must position the stage, and measure the displacement of objects on the stage.

Flow Chart for Optical Trap Software

Modules

  1. Experiment Module, User Input
    • This module will set up the different experiments.
    • It must allow the user to position the stage and use the camera to observe the slide, in preparation of starting a calibration sequence or performing a measurement.
  2. Stage Positioning & Dead Reckoning
    • This module controls the picomotors (stepper motors) by connecting to a DAC motor interface.
    • It must be able to keep track of the stage position relative to a user defined reference point.
  3. Particle Position Readout
    • This module must accept calibration data (from a file or memory) and then readout the photodiode signal as a position vector.
  4. Particle Imaging
    • This module images the slide and presents a real-time readout to the user. It may optionally include visual tracking of slide objects.
  5. Data Logging
    • The results of an experiment run are collected and analyzed, for example by plotting the extension of the DNA tether vs. the applied force (proportional to displacement).

Notes for Tyler and Marjon in summer, 2008

Day 1 (6-17): Run through this lab as if you have had little to no experience in the 111 lab. Take notes on areas that need further clarification. Try to do this without asking any questions about the procedure, though feel free to ask questions about the trapping theory. If you need to ask questions about the procedure because the write up is unclear, write down how so and at the end of the day, shoot me an email with these points and I will fix them in the evening: owenst@berkeley.edu

Day 2 (6-18)

  • I edited the program to allow for better real time viewing, to clarify the graphs and to graph more stuff. Now when you save data for 1/6s and 4/3s it will graph the Vx, Vy positions. Let me know if this causes a problem.
  1. Try to open the Prosilica Viewer program when you DON'T have administrative privelege. Ask Don to take away one of your administrative status's.
  2. Attempt the stuck bead scanning method. I have rewritten this section to make it more clear. It is a rather tricky section and took me awhile to figure out. I think if you just read through the whole thing very carefully and try scanning a few beads, you will eventually get some good data.
  3. Move onto the force calibration methods, taking and analyzing data. Try to do all of this in Excel First. If time permits, at the end of the week we can try out the Matlab stuff....are either of you rather familiar with matlab?
  4. I will be working on another project during the day, but feel free to call me after 10: 510-331-4331

Day 3 (6-19)

  • I edited the program again so that the program now downsamples the data at 30Hz. So in the notepad file created by the 8s @ 1kHz reading, you will now see an X and Y position sampled at 1kHz for 8 seconds and another two columns, with only about 240 points. These are the X and Y position down sampled to 30Hz. This can be used in the equipartition theorem method to estimate the trap stiffness, since these should be roughly independent measurements of the position of the bead. The process is actually very easy, but there is a lot of theory behind it. You don't need to look into this, but you might want to glance at it. I have already researched it quite a bit and don't have much more time to put into it before I go.
  1. Finish the laser bead scanning and fit a line to the linear region of the data to find out the sensitivity (QPD Voltage to distance travelled conversion). This should be roughly the same in the X and Y direction, though vary with bead size. I didn't find this to be the case, which may be a result of wave front of the laser beam being somewhat elliptical or varying in intensity. What do you find?
  2. Continue with the other force calibrations (Equipartition Theorem Method and Rolloff Frequency Method NOT Stokes Drag), taking each of them at say, 3 different powers of the laser: maybe 130, 200 and 270 mA or whatever you want (I am refering the the current delivered to the laser diode, which can be read off of the Laser Diode Controller). This should be quick, since you can trap one bead in a dilute solution away from the glass, take a 4/3s PSD reading and and 8s 1kHz reading at each power: BOOM, done.
  3. Analyze your data and see if the trapping stiffness, k, increases linearly with the laser power. This requires that you know the sensitivity of the trap. If you data is good, k should depend linearly on the laser power within a certain range, increasing as the laser power increases.
  4. I'll come in around 4 to see how things went and to use the trap! If you finish analyzing data early, then you can play with the onion cell or do whatever you want. Maybe you can go buy an ice cream cone 'cuz its gonna be hot!

Day 4 (6-20)

  • I edited the program again. I noticed that Excel seemed to be crashing a lot when Marjon was using it the other day, so I changed the data acquisition rate to a maximum of 12kHz. Now you can just take a lot of data at 12kHz, but no longer at 24kHz. This will half the amount of data you should be throwing around. I also corrected down sampling typo: it says 30 Hz now.
  1. Try to do these first two steps before lunch:
    1. Find the trap stiffness,k, and sensitivity, ρ from the PSD data. Don't do the peak value method. Ya'll have already got some data on this. Remember, only use the lower frequency portion of the PSD data. Throw away the upper half.
    2. Come up with another estimate for trap stiffness using the Equipartition Theorem at the same 3 trapping strengths.
  2. Now, do the biological section, part 2: Trapping E Coli. The most important part here is to observe the tumble. To see this, however, you must have highly mobile E. Coli, so it may take a couple samples.
  3. If time permits, work on the onion cell stuff
    1. I will need quite a bit of commentary on this. Are you able to do the procedures? Are they easy? Suggestions?
  4. I will not be in until later in the afternoon. If I don't make it in in time to see you, could you send me an email with what you thought about things. owenst@berkeley.edu


Good luck...call if you get stuck or talk to Don

-Trevor

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