The overall goal of this procedure is to manipulate short DNA molecules of 100 nanometers in length with constant well calibrated optical forces. This is accomplished by tethering the DNA on one end to a cover slip and the other to a polystyrene microsphere. Next, the optical tweezers are aligned and calibrated.
Then the DNA is stretched away from the surface with a combination of gradient forces and scattering forces acting on the microsphere. Finally, the DNA is extended by trapping the bead in the linear region of the potential. The protocol has been used to study mechanical properties of DNA and DNA protein binding kinetics, and has further been useful in elucidating the importance of boundary conditions on elasticity of short DNA constructs.
Generally, individuals new to this method will struggle because alignment and calibration of the constant force optical tweezers are difficult and complex. The optical tweezers include a beam from a 1064 nanometer laser that is split into two orthogonally polarized beams. One is used to manipulate the biomolecule and the other is used for calibration purposes.
The intensity of the manipulation beam is controlled by an AO optic deflector or a OD.The beams are then recombined with another polarizing beam splitter. The beams are tightly focused by a plan APO 60 x 1.4 oil immersion microscope objective onto the sample cell. This tweezer setup is combined with a home-built brightfield microscope that provides illumination from a halogen lamp focused onto the sample chamber by a condenser.
The bright field image is separated from the laser light by a dichroic mirror and then imaged on two CCD cameras. One CCD acts as our primary means of acquiring data and is software triggered to yield precise sampling rates. While the other CCD is used to image a single stuck reference microsphere, whose position serves as a feedback control system to compensate for drift in the microscope.
The sample is coarsely positioned with respect to the objective by an XY stage, and then can be precisely positioned by an embedded three dimensional piso stage. The forward scattered and transmitted laser light is collected by the bright field condenser and focused onto a photo detector to calibrate the size of the bead, load a sample chamber with a dispersed solution of 800 nanometer diameter polystyrene microspheres diluted in phosphate buffered saline. Let it sit for 10 to 15 minutes and then lightly flush with buffer.
To remove excess microspheres, locate a microsphere randomly stuck to the cover glass and adjust the height of the sample chamber until the microsphere is approximately one micrometer below the focus. To generate a defocused image to measure the apparent size of the image using lab view first, find the center of the microsphere using the geometric pattern matching function. Next, generate a radial intensity profile of the microsphere by averaging 360 degrees.
About the center of each cross section fit the radial profile, which corresponds to a white ring in each of the bright field images with a quadratic function. To find a brightness peak, the distance between this peak and the center can be used as a measure of the apparent size of the bead using the calibrated piso stage gradually increase the axial position of the microsphere and acquire an image at each axial position with the CCD camera. Repeat the image analysis for each successive image to correlate the apparent size of the microsphere with its axial location.
The axial resolution obtained by this method is around 1.4 nanometers to map the optical potential of the manipulation beam begin by coline, aligning the manipulation beam and the calibration beam with the manipulation beam switched off. Confine a free microsphere within the much stiffer trap of the calibration beam. Now turn on the manipulation beam.
Since it is much weaker than the calibration beam, the axial position of the microsphere will be slightly perturbed. The resulting change in the axial position can be measured from the defocused bright field images as already described. Shift axial focus and repeat plot the displacement delta X against the axial position of the focus of the calibration beam.
The axial position corresponding to the largest displacement of the microsphere determines the center of the linear region of the optical trap. For stretching a DNA sample mount the chamber containing surface tethered DNA molecules, and while observing the bright field image, place the focus of the manipulation beam slightly above the unstretched microspheres by adjusting the telescope, then adjust the position of the stage until one of the microspheres is trapped. Roughly position the microsphere in the center of the XY plane of the optical trap, generate a series of square waves in lab view and send them to the A OD to repetitively turn the laser beam on and off.
Watch the microsphere as the trap is repeatedly turned on and off. And note if the laser induces any preferential direction to the microspheres motion while iteratively adjusting the microsphere position in both the X and y direction. By controlling the piso stage, the random motion of the microsphere should become isotropic in the X five plane, although noticeably restricted when the laser is on.
Next, align the bead in the Z direction. Again, pulse the laser beam on and off. While this time simultaneously measuring the microspheres axial displacement in real time.
Center the stage within the linear region, which is the point where the Z displacement is greatest for stretching the DNA ramp, the laser intensity by sending a voltage signal to the A OD from zero volts to 0.5 volts in steps of 0.025 volts in each step. Record 400 frames at 100 frames per second and average them to obtain the axial displacement. Finally, plot the force extension curves and fit them to a modified worm-like chain model.
Shown here are force extension curves for 2D NA sequences, 1, 298 base pairs and 247 base pairs in length for short stretches of DNA. The conventional worm-like chain or WLC model does not fully explain the force extension relationship because at these length scales, one must account for finite size effects and zero force extension arising from boundary constraints. The force extension measurements therefore have to be fit using a modified WLC model, which has an effective persistence length and a zero force extension as fit parameters described further in the supplementary materials.
For large contour lengths of D-S-D-N-A, the effective persistence length is simply the nominal persistence length about 50 nanometers, and the zero force extension can be neglected from the modified WLC fits. The effective persistence lengths are 35 nanometers for the 1, 298 base pair, DNA and 25 nanometers for 247 base pair DNA After its development. This method paved the way for a range of nano mechanical measurements on DNA and DNA protein complexes, including a study of the kinetics of the formation and breakdown of regulatory protein mediated DNA loops.
