Back-focal-plane interferometry is used to measure displacements of optically trapped samples with very high spatial and temporal resolution. However, the technique is closely related to a method that measures the rate of change in light momentum. It has long been known that displacements of the interference pattern at the back focal plane may be used to track the optical force directly, provided that a considerable fraction of the light is effectively monitored. Nonetheless, the practical application of this idea has been limited to counter-propagating, low-aperture beams where the accurate momentum measurements are possible. Here, we experimentally show that the connection can be extended to single-beam optical traps. In particular, we show that, in a gradient trap, the calibration product κ·β (where κ is the trap stiffness and 1/β is the position sensitivity) corresponds to the factor that converts detector signals into momentum changes; this factor is uniquely determined by three construction features of the detection instrument and does not depend, therefore, on the specific conditions of the experiment. Then, we find that force measurements obtained from back-focal-plane displacements are in practice not restricted to a linear relationship with position and hence they can be extended outside that regime. Finally, and more importantly, we show that these properties are still recognizable even when the system is not fully optimized for light collection. These results should enable a more general use of back-focal-plane interferometry whenever the ultimate goal is the measurement of the forces exerted by an optical trap.

Despite the tremendous success of force-measuring optical traps in recent years, the calibration methods most commonly used in the field have been plagued with difficulties and limitations. Force sensing based on direct measurement of light momentum changes stands out among these as an exception. Especially significant is this method's potential for working within living cells, with non-spherical particles or with non-Gaussian beams. However, so far, the technique has only been implemented in counter-propagating dual-beam traps, which are difficult to align and integrate with other microscopy techniques. Here, we show the feasibility of a single-beam gradient-trap system working with a force detection technique based on this same principle.

In optical tweezers, heating of the sample due to absorption of the laser light is a major concern as temperature plays an important role at microscopic scale. A popular rule of thumb is to consider that, at the typical wavelength of 1064 nm, the focused laser induces a heating rate of B = 1 °C/100 mW. We analysed this effect under different routine experimental conditions and found a remarkable variability in the temperature increase. Importantly, we determined that temperature can easily rise by as much as 4 °C at a relatively low power of 100 mW, for dielectric, non-absorbing particles with certain sets of specific, but common, parameters. Heating was determined from measurements of light momentum changes under drag forces at different powers, which proved to provide precise and robust results in watery buffers. We contrasted the experiments with computer simulations and obtained good agreement. These results suggest that this remarkable heating could be responsible for changes in the sample under study and could lead to serious damage of live specimens. It is therefore advisable to determine the temperature increase in each specific experiment and avoid the use of a universal rule that could inadvertently lead to critical changes in the sample.

The well calibrated force‐extension behaviour of single double‐stranded DNA molecules was used as a standard to investigate the performance of phase‐only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 μm particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high‐force capability for holographic optical traps achievable by SLM technologies. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)