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Laser Tweezers Experimental Procedure Ernest Kempton Adams Laboratory The following pages are meant to briefly introduce you to the history of laser tweezers, the theory behind them, and how to use our particular apparatus to make a few of the many measurements possible with this device. They are meant as a primer to optical trapping and as a guide to the idiosyncrasies of our particular setup. However, it is not the intention that you will use this guide as your only source of information on this interesting and useful application of laser light. An extensive list of references -- with brief synopses where necessary -- are included in appendix A. In addition, many of these references, including the book, Laser Tweezers In Cell Biology, ought to accompany the apparatus. Introduction History Staring through a microscope at Bell Labs in New Jersey, Dr. Art Ashkin sat looking through his microscope onto the miniature world below. His sample, which had a laser focused on it, was behaving in a way that he did not expect. To his surprise, he noticed that some of the particles in his sample began to "stick" to the laser beam and follow it around as the laser tracked across the field of view. After further investigation, it was clear that for some reason the laser beam was in fact acting like a pair of microscopic tweezers, squeezing particles at the point of its focus and holding them stationary while the rest of the sample moved about it. This accidental discovery led to a paper published by Ashkin's group in 1986, reporting the achievement of "the first experimental observation... of a single-beam gradient force radiation pressure particle trap." This serendipitous development would lead to an entirely new technique for manipulating microscopic and sub-microscopic particles, eventually being applied to individual atoms. Due to their ability to precisely manipulate microscopic particles, laser tweezers lend themselves to a wide array of different applications in experimental physics, biology and chemistry. Objects that are smaller than ten microns-a human hair is 100 microns thick-can be trapped and moved about. In addition, because the shape of the potential energy well in which the particle is trapped can be well characterized, measuring forces on the order of piconewtons is possible. To get a sense of the scale of a piconewton, the force on a kilogram mass on the Earth’s surface is roughly 10 newtons, while a piconewton is one trillionth of this. These two abilities, manipulation and force measurement, can be used to accomplish many novel procedures. One such procedure results from the accessibility of lasers to spaces that are unreachable with solid instruments. Most devices that dexterously manipulate matter require a solid handle to be attached to the grasping tip, a forklift or a set of forceps, for example. Laser tweezers require only a transparent through which a laser beam can travel. A potent illustration of this advantage is seen in the manipulation of organelles within the confines of a living cell. Light from the laser passes through the cell wall and only affects the organelle that it is focused on. In this way, properties of internal structure of living organisms can be explored. Laser tweezers have also been used by scientists to measure the forces on microscopic particles. Although many biological substances cannot be trapped directly, they can be attached to particles that are easily trapped. A molecule can be stretched out like springs between a solid anchor and the particle controlled by the laser. The molecule's stiffness can be calculated by observing the distance out of the center of the trap traveled by the entrapped particle when pulled out of the focal point of the laser by another force, which could arise from either a second optical trap or the attachment of part of the object to a fixed surface. The springiness of DNA and elastic muscular proteins are measured in this way. Motile cells such as sperm and flagellants can be tested to determine the strength of their swimming under different conditions. This is noted by observing their ability to swim out of traps of varying strengths. Sperm are subjected to varied temperatures and environmental chemistry in order to find the ideal conditions for insemination. Related to reproduction, artificial insemination can be aided by the placement of sperm cells in the proximity of an egg in order to increase the chance of conception. Multiple lasers focused on the same sample can be used to manipulate several different objects simultaneously. Two cells can be brought together and, after their membranes are cut open, fused into a single hybrid cell. In addition, arrays of tweezers can be used to create complex three-dimensional structures out of molecules that can then be frozen into place. This technique is currently being developed and may lead to the ability to mass-produce micro devices with useful electronic and mechanical properties. Current uses Laser tweezers continue to be of great use in basic research. Due to advances in diode laser technology, it is now possible to access powerful lasers in a variety of wavelengths at lower costs than ever before. The laser tweezers is a very useful tool for biophysicists who wish to measure the miniscule forces exerted by the cellular interactions in vivo, an important area of current research. With the availability of a large variety of commercially available protein coated polystyrene beads, researchers are able to probe interactions between the cell and its external environment with precision using this very flexible experimental technique. A search of current scientific journals will yield a panoply of other current applications. This setup The purpose of the apparatus and this procedure is to familiarize you with the basic theory and function of laser trapping. Though research grade laser traps are generally much more powerful (and expensive), the apparatus you will be working with is, delicate, costly, potentially dangerous, and can perform many of the same tasks as more elaborate setups. Caution should be taken when working with this apparatus both when the laser in on as well as when it is off. A slight perturbation to the optical path can misalign the laser and require a tedious and time-consuming period of realignment. The laser that traverses this optical path has a power of close to 30mW and is carried in the form of 633 nm (red) photons that are readily absorbed by the retina. Thus, in the best interest of your eyes, you must wear goggles when the primary laser is operating. The moment you relax around the laser is the moment that an accident will happen, no matter how comfortable you may feel. Figure 1 Figure 2 Getting Familiar With the Apparatus Optical Path In order to familiarize you with the apparatus, start at the source of the optical path, the laser itself. The laser uses He-Ne gas to produce a coherent emission that is capable of a maximum output of 30mW at 632.8nm. The laser is attached to its power supply that is turned on via a key. The next elements in the optical path are two mirrors. These mirrors serve to redirect the laser beam. These mirrors allow us to steer the laser beam at any angle and at any height (within the confines of the range of much of the mirrors). The beam is steered by these mirrors into a set of two lenses. These lenses act as a telescope, increasing the diameter of the beam for reasons that will be explained later. Figure 3 After the beam passes through the telescoping lenses, it is vertically redirected by a third mirror. This mirror serves to send the beam through a third lens that will give the beam front the optimal radius of curvature. The beam then strikes a fourth mirror and is redirected into a side port that is bored into the side of the aluminum adapter sitting on top of the microscope. The adapter holds two dichroic reflectors. These act as mirrors for long wavelength light while being transparent for light of higher frequencies. Thus, when the red laser beam meets the dichroic surface it reflects down the barrel of the microscope where it then meets the back of the microscope objective. The use of the lenses in the beam path previously discussed is entirely based on the properties of the objective. The telescope is de-signed to create a beam diameter that matches the diameter of the back of the objective. The result is that all of the laser light enters the objective so that no power is lost and that the laser light entirely fills the back of the objective, allowing the area of glass to be utilized in re-solving the smallest spot possible. The focusing power of the objective is further maximized because the third lens has created a beam front with a radius of curvature ideal for the objective. The objective creates a highly focused spot that will act as our optical trap once a suitable sample is placed on the microscope stage. Of course, the microscope objective continues to focus light in the other direction as well, sending it up the microscope barrel. This light once again passes through the first dichroic reflector. Here the dichroic serves to weaken the intense laser beam so that our detectors are not damaged. The image from the microscope continues upward through the aluminum adapter, striking a second dichroic before reaching the camera. The reflected laser light from this second dichroic will eventually exit the upper side port and be absorbed by a photodiode array that will indicate small changes in a trapped particles position. Procedures Rough Alignment of the Trap: All coarse alignment is to be done with a secondary, less powerful laser source, or by filtering out some of the intensity of the present laser. 1. Align laser path without lenses in place. a. Rotate microscope objective holder until there is no objective in the optical path. If there are no empty holes, carefully unscrew one of the objectives from the microscope. b. Open both apertures in the microscope condenser unit. Check that the apertures are open by turning on microscope illumination and verifying the maximum amount of light passes through condenser. Place a square of paper beneath the condenser, resting on the foot of the microscope, covering the illumination window. c. Roughly align mirrors by unfastening thumbscrews in post bases and rotating the posts until the laser beam reflects off of each mirror down the optical path. Align the dichroic assembly by unscrewing its fastening thumbscrew and rotating the assembly. You should see laser light on the paper beneath the condenser. d. Using fine adjustment screws on mirror mounts, steer the path so that you maximize the amount of laser light passing through the condenser and falling on the paper. If necessary, repeat step c) such that the laser is reflected from the centers of the mirrors in order to allow maximum adjustment flexibility in later steps. e. Place spare posts identical to those of the lenses into the lens post holders. Essentially you mimic adding the lenses, by adding lens posts without a lens. f. By adjusting the height of the lens posts, ensure that the laser beam passes directly across the center of each post. This ensures that when you add the lenses the laser beam can be made to travel directly through the center of each lens. Repeat steps c), d), and f) as necessary. g. Now, close the condenser apertures all the way down. If the beam is precisely aligned, you should still see the laser spot brightly. h. Finally, check that the beam is perpendicular to the stage by holding a blank microscope slide flat on the stage in the path of the laser beam. By tilting the slide slightly, you should be able to find the reflection on mirror 4. If, when the slide lies flat on the stage the reflected beam strikes the mirror at the same spot as the incident beam, the beam is perpendicular with the stage. 2. Add lenses a. Mark the position of the spot that your now well-aligned laser beam makes on the paper below the condenser. b. Remove the bare post from lens 1 position and replace it with a post with the appropriate lens attached. c. Adjust the lens position until the spot on the paper returns to the spot you marked in 2a). d. Repeat steps 2b) and 2c) for lens 2 and lens 3. 3. Verify rough alignment through video system. a. Switch on monitor and video camera. Adjoin the camera to the system by placing the camera’s lens cylinder into the cylindrical opening at the top of the dichroic assembly. b. Turn on microscope illumination. Place a blank slide in microscope stage slide holder. Using a low power objective, focus the microscope onto the top surface of the slide. You may need to mark on the slide with a marker in order to have a reflective surface onto which you can focus. c. You should see the reflection of the laser from the surface of the glass slide. Verify that this reflection is from the laser by slightly adjusting the laser beam using the fine adjustments on mirror 4. Procedure for Preparing a Bead Sample: 1. 2. 3. 4. 5. 6. 7. 8. Using a glass transfer pipette and a rubber bulb, measure out 10 ml of distilled water into a graduated cylinder. For a very dilute sample, measure 15-30 ml of distilled water. For a more concentrated sample of beads, use less distilled water. From the refrigerator, take a dropper bottle containing the appropriately sized beads. Shake the dropper bottle until the beads enter a suspension. There should be no clumps of beads and the suspension should resemble milk. If shaking the bottle does not provide adequate agitation, you may need to remove the dropper bottle’s cap and use a pipette to repeatedly take up and expel the contents of the bottle until the suspension becomes homogeneous. Squeeze a single drop of the bead suspension into the distilled water. Using the pipette, take up and expel the distilled water until the new, dilute suspension is thoroughly mixed. Taking a clean cover slip and using the vacuum grease filled syringe, form a thin but continuous border of grease along the edges of a cover slip on one side. Lay the cover slip down, grease side facing up. Using a transfer pipette, add enough of your diluted bead suspension to the grease well on the cover slip, so that the suspension covers the bottom inside of the well. Place a clean microscope slide on top of the cover slip. This should be done in such a way that the grease on the cover slip makes a seal against the slide but does not seal until all the air inside is expelled. This is done in order to prevent bubbles from being present in your sample. Pick up your slide and flip it over. Using a wipe, dry any suspension that was expelled from the well in step 7. Procedure for Measuring the Maximum Trapping Strength: In order to empirically determine the maximum strength of your laser trap you will utilize an equation that gives a simple description of the viscous drag force that acts between a bead held stationary by the laser tweezers and the surrounding fluid in your sample. This equation, called the Stokes drag equation, is of the form: F = 6 πηrv, where η is the viscosity of the water, which is approximately 10-3 Ns/m2, v is the escape velocity of the bead from the trap and r is the radius of the sphere. The Stokes equation will not hold if the bead is less than several bead diameters from the top of the cover slip due to surface effects that affect the viscous drag. In addition, spherical aberrations will drastically reduce trapping strength with increasing depths. Therefore, all measurements of the trapping strength should be done at a few diameters below the surface. Recording the Bead Escape Velocity: 1. 2. 3. 4. 5. 6. Align the laser tweezers following the appropriate procedure. Prepare a sample of beads following the appropriate procedure. Place the slide, cover slip up, in the microscope sample holder. Trap a single bead just below the cover slip surface. Turn on the VCR system and begin recording. Translate the stage with a slight acceleration until the bead falls out of the laser trap. 7. Stop recording. Make a note of the tape position and record the diameter of the beads used for this measurement. 8. Repeat steps 2-7 for differently sized beads. Analyzing the Bead Escape Velocity: 1. Advance the tape to the point just before the bead escapes from the trap and, using the VCR remote control, press the pause button. 2. Using the frame advance button of the VCR, measure the distance that an object that is affixed to the cover glass (a stuck bead, a piece of dust, etc.) moves with each depression of the advance button. Calibrating Distance Scales: Distance scales for the video system are easily found by noting the resulting image size for a bead of known diameter. Calibrating Time Scales: Time elapsed with each depression of the frame advance is 1/30 of a second. This can be verified by making a recording of an image that changes regularly with a known period and than counting the number of frame advance depressions required to advance through an entire period. Making a recording of the second hand of a watch is one such example.