Optical traps are commonly constructed with high-numerical-aperture objectives. Oil-immersion objectives suffer from spherical aberrations when used for imaging in aqueous solutions. The effect of spherical aberrations on trapping strength has been modeled by approximation, and only a few experimental results are available in the case of micrometer-sized particles. We present an experimental study of the dependence of lateral and axial optical-trap stiffness on focusing depth for polystyrene and silica beads of 2 m diameter by using oil-and water-immersion objectives. We demonstrate a strong depth dependence of trap stiffness with the oil-immersion objective, whereas no depth dependence was observed with the water-immersion objective.

The micrometer-scale length of some protein polymers allows them to be mechanically manipulated in single-molecule experiments. This provides a direct way to measure persistence length. We have used a double optical trap to elastically deform single microtubules and actin filaments. Axial extensional force was exerted on beads attached laterally to the filaments. Because the attachments are off the line of force, pulling the beads apart couples to local bending of the filament. We present a simple mechanical model for the resulting highly nonlinear elastic response of the dumbbell construct. The flexural rigidities of the microfilaments that were found by fitting the model to the experimentally observed force-distance curves are (7.1 +/- 0.8) x 10(4) pN nm2 (persistence length L(p) = 17.2 microm) for F-actin and (6.1 +/- 1.3) x 10(6) pN nm2 (L(p) = 1.4 mm) for microtubules.

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Displacements of optically trapped particles are often recorded using back-focal-plane interferometry. In order to calibrate the detector signals to displacements of the trapped object, several approaches are available. One often relies either on scanning a fixed bead across the waist of the laser beam or on analyzing the power spectrum of movements of the trapped bead. Here, we introduce an alternative method to perform this calibration. The method consists of very rapidly scanning the laser beam across the solvent-immersed, trapped bead using acousto-optic deflectors while recording the detector signals. It does not require any knowledge of solvent viscosity and bead diameter, and works in all types of samples, viscous or viscoelastic. Moreover, it is performed with the same bead as that used in the actual experiment. This represents marked advantages over established methods.

In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of doublestranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.