3 research outputs found
Calibration of optical tweezers with positional detection in the back-focal-plane
We explain and demonstrate a new method of force- and position-calibration
for optical tweezers with back-focal-plane photo detection. The method combines
power spectral measurements of thermal motion and the response to a sinusoidal
motion of a translation stage. It consequently does not use the drag
coefficient of the trapped ob ject as an input. Thus, neither the viscosity,
nor the size of the trapped ob ject, nor its distance to nearby surfaces need
to be known. The method requires only a low level of instrumentation and can be
applied in situ in all spatial dimensions. It is both accurate and precise:
true values are returned, with small error-bars. We tested this experimentally,
near and far from surfaces. Both position- and force-calibration were accurate
to within 3%. To calibrate, we moved the sample with a piezo-electric
translation stage, but the laser beam could be moved instead, e.g. by
acousto-optic deflectors. Near surfaces, this precision requires an improved
formula for the hydrodynamical interaction between an infinite plane and a
micro-sphere in non-constant motion parallel to it. We give such a formula.Comment: Submitted to: Review of Scientific Instruments. 13 pages, 5 figures.
Appendix added (hydrodynamically correct calibration
Stepwise bending of DNA by a single TATA-box Binding Protein
The TATA-box Binding Protein (TBP) is required by all three eukaryotic RNA
polymerases for the initiation of transcription from most promoters. TBP
recognizes, binds to, and bends promoter sequences called ``TATA-boxes'' in the
DNA. We present results from the study of individual Saccharomyces cerevisia
TBPs interacting with single DNA molecules containing a TATA-box. Using video
microscopy, we observed the Brownian motion of beads tethered by short
surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the
DNA changes and the amplitude of Brownian motion of the tethered bead is
reduced compared to that of unbent DNA. We detected individual binding and
dissociation events and derived kinetic parameters for the process.
Dissociation was induced by increasing the salt concentration or by directly
pulling on the tethered bead using optical tweezers. In addition to the
well-defined free and bound classes of Brownian motion, we observed another two
classes of motion. These extra classes were identified with intermediate states
on a three-step, linear binding pathway. Biological implications of the
intermediate states are discussed.Comment: Accepted for publication in: Biophysical Journa