Juleon M. Schins

Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.

Three dimensional single-particle tracking with nanometer resolution

We have developed a method for three dimensional (3D) tracking of polystyrene spheres with nanometer resolution. The detection technique is based on measuring the displacement of a polystyrene sphere positioned in the center of a laser beam just behind the focus. A change in the lateral position of the sphere causes a deflection of the beam which can be measured using a position sensitive detector. A change in the axial position of the sphere causes a shift in the axial position of the focus behind another lens, which can be measured using an overfilled photodiode. A feedback system is used to keep the sphere in the center of the laser beam to avoid the influence of lateral displacements on the detection of the axial position. Spatial resolution for a 0.92 μm polystyrene sphere was better than 1 nm in three dimensions using a sampling rate of 1 kHz. This method was applied to track spheres bound to adhesion molecules LFA-1 expressed at the surface of living cells. It turned out to be a useful method to ac...

Polarization effects in flow cytometric DNA sizing

We have investigated the influence of the polarization direction of excitation light on DNA sizing results obtained by a flow cytometric technique. We found strong fluorescence anisotropy of the fluorescent signal from lambda DNA stained with the bis-intercalating dye TOTO-1. Small fragments of DNA are less sensitive to polarization than larger pieces. This effect is more pronounced at faster flow speeds. These observations show a noticeable orientation of the DNA molecules introduced by the shear forces in the flow system. The data are consistent with an angle between the transition moment of fluorescence of TOTO-1, and the long axis of DNA is approximately 62 degrees .

Photon-counting device compatible with conventional flow cytometric data acquisition electronics

We present an electronic scheme that enables us to use a photon-counting device (photomultiplier or avalanche photodetector) for measuring extremely weak signals in a flow cytometer. It can be used as a sole detector, or in combination with other (conventional) detectors using the data acquisition hardware of a conventional flow cytometer. The essential principle is that photon-counting pulses are converted to an analogue signal that is continuously proportional to the number of detected photons during the last integration time. The integration time should be approximately equal to the time an object is illuminated in the flow chamber. In this way, the photon burst due to real events is measured correctly and discriminated from the background pulses (fluorescence and Raman). The use of this scheme for the measurement of single DNA molecules is illustrated.

New technique for high resolution DNA sizing in epi-illumination

We present a high-resolution DNA-sizing technique based on the principles of flow cytometry, using a high numerical aperture objective and epi-illumination. The new technique, designed for small fluorescing samples/particles (sub-micron diameter) suspended in a weakly fluorescent medium, makes use of an additional focus for high-precision particle localisation. This way, only those particles are considered that flow exactly through a well-defined volume. Results are presented for fluorescent beads, as well as for YOYO-stained plasmids containing 5,500 basepairs. The latter were measured with 6.2% resolution, setting a new limit to flow-based sizing of DNA.

Mathematics: A Pointer to an Independent Reality

It is an irony of history that Alan Turing actually offered the main ingredient for an elegant proof of the non-algorithmic nature of human understanding [1]. Turing has always been convinced that human intellect, though (sometimes) more powerful than computers, is not essentially superior [2], Yet his argument is not very convincing, and does not stand the more recent critique of Roger Penrose [3].

Quantum Theory: A Pointer to an Independent Reality

Quite soon after the first predictions of quantum mechanics the founding fathers became aware of two major promises contained in this new theory: first, it seemed to have an exceptional predictive power in many physical and chemical disciplines; second, it would launch, like no other physical theory before, an equally exceptional philosophical discussion concerning the status of reality. The fundamental issue in this philosophical debate is whether things are, on quantum level, the same way as they are on a classical level.