Artifact-free holographic light shaping through moving acousto-optic holograms.

Holographic light modulation is the most efficient method to shape laser light into well-defined patterns and is therefore the means of choice for many intensity demanding applications. During the last two decades, spatial light modulators based on liquid crystals prevailed among several technologies and became the standard tool to shape light holographically. But in the near future, this status might be challenged by acousto-optic deflectors. These devices are well known for their excelling modulation rates and high optical power resilience. But only few scattered precedents exist that demonstrate their holographic capabilities, despite the many interesting properties that they provide. We implemented a holographic acousto-optic light modulation (HALM) system, that is based on displaying holograms on acousto-optic deflectors. We found that this system can eliminate the ubiquitous coherent artifacts that arise in holography through the inherent motion of acousto-optic holograms. That distinguishes our approach from any other holographic modulation technique and allows to reconstruct intensity patterns of the highest fidelity. A mathematical description of this effect is presented and experimentally confirmed by reconstructing images holographically with unprecedented quality. Our results suggest that HALM promotes acousto-optic deflectors from highly specialized devices to full-fledged spatial light modulators, that can compete in a multitude of applications with LC-SLMs. Especially applications that require large optical output powers, high modulation speeds or accurate gray-scale intensity patterns will profit from this technology. We foresee that HALM may play a major role in future laser projectors and displays, structured illumination microscopy, laser material processing and optical trapping.

Positioning Accuracy in Holographic Optical Traps.

Spatial light modulators (SLMs) have been widely used to achieve dynamic control of optical traps. Often, holographic optical tweezers have been presumed to provide nanometer or sub-nanometer positioning accuracy. It is known that some features concerning the digitalized structure of SLMs cause a loss in steering efficiency of the optical trap, but their effect on trap positioning accuracy has been scarcely analyzed. On the one hand, the SLM look-up-table, which we found to depend on laser power, produces positioning deviations when the trap is moved at the micron scale. On the other hand, phase quantization, which makes linear phase gratings become phase staircase profiles, leads to unexpected local errors in the steering angle. We have tracked optically trapped microspheres with sub-nanometer accuracy to study the effects on trap positioning, which can be as high as 2 nm in certain cases. We have also implemented a correction strategy that enabled the reduction of errors down to 0.3 nm.

Influence of experimental parameters on the laser heating of an optical trap.

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.

Extending calibration-free force measurements to optically-trapped rod-shaped samples.

Optical trapping has become an optimal choice for biological research at the microscale due to its non-invasive performance and accessibility for quantitative studies, especially on the forces involved in biological processes. However, reliable force measurements depend on the calibration of the optical traps, which is different for each experiment and hence requires high control of the local variables, especially of the trapped object geometry. Many biological samples have an elongated, rod-like shape, such as chromosomes, intracellular organelles (e.g., peroxisomes), membrane tubules, certain microalgae, and a wide variety of bacteria and parasites. This type of samples often requires several optical traps to stabilize and orient them in the correct spatial direction, making it more difficult to determine the total force applied. Here, we manipulate glass microcylinders with holographic optical tweezers and show the accurate measurement of drag forces by calibration-free direct detection of beam momentum. The agreement between our results and slender-body hydrodynamic theoretical calculations indicates potential for this force-sensing method in studying protracted, rod-shaped specimens.

A monolithic glass chip for active single-cell sorting based on mechanical phenotyping.

The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications.

Dynamic operation of optical fibres beyond the single-mode regime facilitates the orientation of biological cells.

The classical purpose of optical fibres is delivery of either optical power, as for welding, or temporal information, as for telecommunication. Maximum performance in both cases is provided by the use of single-mode optical fibres. However, transmitting spatial information, which necessitates higher-order modes, is difficult because their dispersion relation leads to dephasing and a deterioration of the intensity distribution with propagation distance. Here we consciously exploit the fundamental cause of the beam deterioration-the dispersion relation of the underlying vectorial electromagnetic modes-by their selective excitation using adaptive optics. This allows us to produce output beams of high modal purity, which are well defined in three dimensions. The output beam distribution is even robust against significant bending of the fibre. The utility of this approach is exemplified by the controlled rotational manipulation of live cells in a dual-beam fibre-optical trap integrated into a modular lab-on-chip system.

Holographic optical tweezers combined with back-focal-plane displacement detection.

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.

Positional stability of holographic optical traps.

The potential of digital holography for complex manipulation of micron-sized particles with optical tweezers has been clearly demonstrated. By contrast, its use in quantitative experiments has been rather limited, partly due to fluctuations introduced by the spatial light modulator (SLM) that displays the kinoforms. This is an important issue when high temporal or spatial stability is a concern. We have investigated the performance of both an analog-addressed and a digitally-addressed SLM, measuring the phase fluctuations of the modulated beam and evaluating the resulting positional stability of a holographic trap. We show that, despite imparting a more unstable modulation to the wavefront, our digitally-addressed SLM generates optical traps in the sample plane stable enough for most applications. We further show that traps produced by the analog-addressed SLM exhibit a superior pointing stability, better than 1 nm, which is comparable to that of non-holographic tweezers. These results suggest a means to implement precision force measurement experiments with holographic optical tweezers (HOTs).

Adding functionalities to precomputed holograms with random mask multiplexing in holographic optical tweezers.

In this study, we present a method designed to generate dynamic holograms in holographic optical tweezers. The approach combines our random mask encoding method with iterative high-efficiency algorithms. This hybrid method can be used to dynamically modify precalculated holograms, giving them new functionalities-temporarily or permanently-with a low computational cost. This allows the easy addition or removal of a single trap or the independent control of groups of traps for manipulating a variety of rigid structures in real time.

Correction of aberration in holographic optical tweezers using a Shack-Hartmann sensor.

Optical aberration due to the nonflatness of spatial light modulators used in holographic optical tweezers significantly deteriorates the quality of the trap and may easily prevent stable trapping of particles. We use a Shack-Hartmann sensor to measure the distorted wavefront at the modulator plane; the conjugate of this wavefront is then added to the holograms written into the display to counteract its own curvature and thus compensate the optical aberration of the system. For a Holoeye LC-R 2500 reflective device, flatness is improved from 0.8λ to λ/16 (λ=532 nm), leading to a diffraction-limited spot at the focal plane of the microscope objective, which makes stable trapping possible. This process could be fully automated in a closed-loop configuration and would eventually allow other sources of aberration in the optical setup to be corrected for.

Fast generation of holographic optical tweezers by random mask encoding of Fourier components.

The random mask encoding technique of multiplexing phase-only filters can be easily adapted to the generation of holographic optical tweezers. The result is a direct, non-iterative and extremely fast algorithm that can be used for computing arbitrary arrays of optical traps. Additional benefits include the possibility of modifying any existing hologram to quickly add more trapping sites and the inexistence of ghost traps or replicas.

Excised bone structures in mice: imaging at three-dimensional synchrotron radiation micro CT.

Bone microarchitecture and mineralization were determined at three-dimensional synchrotron radiation micro computed tomography in two inbred mice strains. Distal metaphysis of the left femur was imaged in three dimensions at 6.65 microm, whereas the right femur was analyzed with histomorphometry. Three-dimensional quantitative parameters of trabecular and cortical bone architecture were computed. C3H/HeJ@Ico mice had greater bone density and thicker trabeculae; greater cortical bone density, cortical thickness, and porosity; and greater mineralization than did C57BL/6J@Ico mice. The technique is well suited for assessment of trabecular and cortical bone in small animals and at the same time provides mineralization status in three dimensions.