Heikki Ojala

Stiffer optical tweezers through real-time feedback control

Using real-time re-programmable signal processing we connect acousto-optic steering and back-focal-plane interferometric position detection in optical tweezers to create a fast feedback controlled instrument. When trapping 3μm latex beads in water we find that proportional-gain position-clamping increases the effective lateral trap stiffness ∼13-fold. A theoretical power spectrum for bead fluctuations during position-clamped trapping is derived and agrees with the experimental data. The loop delay, ∼19μs in our experiment, limits the maximum achievable effective trap stiffness.

Optical position clamping with predictive control

We increase the effective stiffness of optical tweezers by position clamping a polystyrene bead with a predictive feedback control algorithm. This algorithm mitigates the effect of feedback loop delay. Hence, higher gain than with proportional control can be employed, which results in higher effective trap stiffness, without trap instability. In experiments (initial trap stiffness 0.056 pN/nm with a 1.78 μm diameter polystyrene bead), predictive control increased the effective trap stiffness by 55% relative to proportional control. We also derive theoretical expressions for the power spectra of the bead position controlled by our algorithm.

Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0-100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.

Real-time control of optical tweezers

Optically trapped microshperes can be manipulated by steering the trap beam, while the object position is measured with sub-nanometer resolution. A fast steering system is required to create feedback loop for measurements at a constant force or to increase position detection precision by trap stiffening. Using a real-time re-programmable digital signal processor, we combine steering and position detection to create a fast and versatile closed-loop feedback controlled instrument. We describe the construction and calibration of the instrument. We show that a proportional gain position-clamp algorithm can achieve about 10-fold increase in effective trap stiffness while higher gains lead to unwanted resonances.

High-resolution optical tweezers for investigating DNA-binding/translocating molecular motors

A double-trap optical tweezers instrument was constructed and its spatial resolution measured. The instrument features real-time control that allows feedback based position- and force-clamping experiments. To study RNA-polymerization by QDE-1, an RNA-dependent RNA-polymerase, we tethered a 7250 nt single-stranded DNA molecule between two optically trapped microspheres. Preliminary constant-force extension trajectories and force-extension curves were collected.

Single Molecule Studies on the RNA Polymerase QDE-1 by Optical Tweezers

Cellular regulatory mechanisms which rely on small dsRNA molecules (RNA silencing) are major (new) discoveries in biology.In many eukaryotic organisms silencing is achieved post-transcriptionally through pathways where dsRNA is synthesized by RNA-dependent RNA polymerases (RdRPs) and processed into 21-25 nucleotide long RNA molecules.Here we study QDE-1, an RdRP involved in the quelling (RNA silencing) pathway of Neurospora crassa. This filamentous fungus displays noticeable genomic stability, which has been attributed to quelling and other silencing mechanisms. Recently it was shown that QDE-1 is more active as a DNA-dependent RNA polymerase (DdRP) than as an RdRp and that it is also involved in DNA damage response (1,2). Recombinant QDE-1 displays five different enzymatic activities in vitro (3). The structural and biochemical data on this enzyme is extensive (1-5).We are interested on the basic biophysical parameters of QDE-1 action. The approaches taken rely on single molecule assays using high resolution optical tweezers, combined with fluorescence imaging. An optically levitated ‘dumbbell’ assay is used: the nucleic acid (NA) construct features one or two biotinylated ends that tether two different microspheres. The protein of interest is attached to the microsphere or directly to the NA tether. Since in our double-trap instrument one trap is stable whereas the other mobile, we can manipulate the tethers, detect changes in tether length and stiffness, and apply different forces and simultaneously observe the mobility of the fluorescently labelled template or protein.1. Lee, H-C et al. (2009). Nature 459(7244), 274-277.2. Lee, H-C et al. (2010). PLoS Biol. 8 (10), e1000469.3. Aalto et al. (2010). The Journal of Biological Chemistry, 285, 29367-29374.4. Salgado et al. (2006). PLoS. Biol. 4 (12), e434.5. Makeyev E.V. & Bamford D.H. (2002). Mol Cell 10(6), 1417-1427.