Biomedical subjects

J M Schins

Publications and source records attributed to J M Schins.

4 recordsLinked to original sources

PubMed1999

Orientation of the chromophore dipoles in the TOTO-DNA system.

BACKGROUND: Flow cytometry has been applied successfully to the sizing of medium to large-sized DNA molecules, thanks to the excellent staining properties of cyanine chromophores such as TOTO (homodimer of thiazole orange) (Petty et al.: Anal Chem 67:1755-1761, 1995). The hydrodynamic flow, used to focus the sample molecules in a small laser-illuminated volume, is also responsible for their alignment, thereby allowing the determination of the TOTO-dipole orientation with respect to the DNA axis (Agronskaia et al.: Appl Opt 38:714-719, 1999). METHODS: We present model calculations of the fluorescence yield of TOTO-stained DNA measured in a flow-cytometric setup with high numerical aperture. The models consider different orientations of the chromophore dipoles. RESULTS: Comparison of measurement and calculation suggests that the absorption dipoles of the TOTO molecule make a mean angle of 61 degrees with the helix axis of the DNA molecule. This mean angle can be the consequence of two binding modes. CONCLUSIONS: Our results indicate that any model with a significant contribution of perpendicularly-oriented chromophores fails to reproduce the experimental results.

Bacteriophage lambda↗

PubMed1999

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.

Bacteriophage lambda↗

PubMed1998

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.

DNA↗

PubMed1998

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.

DNA↗