Cortical contractility triggers a stochastic switch to fast amoeboid cell motility.

3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.

Efficient and accurate intensity diffraction tomography of multiple-scattering samples.

Optical Diffraction Tomography (ODT) is a label-free method to quantitatively estimate the 3D refractive index (RI) distributions of microscopic samples. Recently, significant efforts were directed towards methods to model multiple-scattering objects. The fidelity of reconstructions rely on accurately modelling light-matter interactions, but the efficient simulation of light propagation through high-RI structures over a large range of illumination angles is still challenging. Here we present a solution dealing with these problems, proposing a method that allows one to efficiently model the tomographic image formation for strongly scattering objects illuminated over a wide range of angles. Instead of propagating tilted plane waves we apply rotations on the illuminated object and optical field and formulate a new and robust multi-slice model suitable for high-RI contrast structures. We test reconstructions made by our approach against simulations and experiments, using rigorous solutions to Maxwell's equations as ground truth. We find the proposed method to produce reconstructions of higher fidelity compared to conventional multi-slice methods, especially for the challenging case of strongly scattering samples where conventional reconstruction methods fail.

Generally Applicable Holographic Torque Measurement for Optically Trapped Particles.

We present a method to measure the optical torque applied to particles of arbitrary shape such as micrometer-sized micro-organisms or cells held in an optical trap, inferred from the change of angular momentum of light induced by the particle. All torque components can be determined from a single interference pattern recorded by a camera in the back focal plane of a high-NA condenser lens provided that most of the scattered light is collected. We derive explicit expressions mapping the measured complex field in this plane to the torque components. The required phase is retrieved by an iterative algorithm, using the known position of the optical traps as constraints. The torque pertaining to individual particles is accessible, as well as separate spin or orbital parts of the total torque.

Diffractive tunable lens for remote focusing in high-NA optical systems.

Remote focusing means to translate the focus position of an imaging system along the optical axis without moving the objective lens. The concept gains increasing importance as it allows for quick 3D focus steering in scanning microscopes, leaves the sample region unperturbed and is compatible with conjugated adaptive optics. Here we present a novel remote focusing approach that can be used in conjunction with high numerical aperture optics. Our method is based on a pair of diffractive elements, which jointly act as a tunable auxiliary lens. By changing the mutual rotation angle between the two elements, we demonstrate an axial translation of the focal spot produced by a NA = 0.95 air objective (corresponding to NA = 1.44 for an oil immersion lens) over more than 140 µm with largely maintained focus quality. We experimentally show that for the task of focus shifting, the wavefront produced by the high-NA design is superior to those produced by a parabolic lens design or a regular achromatic lens doublet.

Model-based compensation of pixel crosstalk in liquid crystal spatial light modulators.

Spatial light modulators (SLMs) based on liquid crystals are widely used for wavefront shaping. Their large number of pixels allows one to create complex wavefronts. The crosstalk between neighboring pixels, also known as fringing field effect, however, can lead to strong deviations. The realized wavefront may deviate significantly from the prediction based on the idealized assumption that the response across a pixel is uniform and independent of its neighbors. Detailed numerical simulations of the SLM response based on a full 3D physical model accurately match the measured response and properly model the pixel crosstalk. The full model is then used to validate a simplified model that enables much faster crosstalk evaluation and pattern optimization beyond standard performance. General conclusions on how to minimize crosstalk in liquid crystal on silicon (LCoS) SLM systems are derived, as well as a readily accessible estimation of the amount of fringing in a given SLM.

Acoustic force spectroscopy.

Force spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Here we extend the force spectroscopy toolbox with an acoustic manipulation device that can exert forces from subpiconewtons to hundreds of piconewtons on thousands of biomolecules in parallel, with submillisecond response time and inherent stability. This method can be readily integrated in lab-on-a-chip devices, allowing for cost-effective and massively parallel applications.

Multi-color operation of tunable diffractive lenses.

Rotationally tunable diffractive optical elements (DOEs) consist of two stacked diffractive optical elements which are rotated with respect to each other around their central optical axis. The combined diffractive element acts as a highly efficient diffractive lens, which changes its optical power as a function of the mutual rotation angle. Here we show that the principle can be extended to produce polychromatic tunable lenses, i.e. lenses which have the same optical power, and the same diffraction efficiency within the full tuning range at three or more selectable wavelengths. The basic principle is to use higher order DOEs, which will be polychromatic at harmonics of a fundamental wavelength. The method can be applied to other types of optical elements which are tunable by rotation, like axicons, or generalized lenses with arbitrary radial phase profiles, or to elements tunable by a mutual translation, like diffractive Alvarez lenses.

Programmable freeform optical elements.

Modern liquid crystal spatial light modulators (SLMs) are capable of shifting the optical path length by some microns, which corresponds to phase shifts of several multiples of 2π. We use this capability to display freeform optical elements (FOEs) on a SLM, as largely smooth phase variations with only a small number of wrapping lines. These FOEs can be programmed to generate so-called caustic intensity distributions, which may be real images reconstructed at a selected position in front of the SLM surface. In contrast to standard diffractive structures, reconstruction of the freeform images is non-dispersive (i.e. white light images can be programmed), free of speckle, and its efficiency does not depend on the wavelength. These features promise novel applications in image projection, and various application fields of SLMs in microscopy.

Modified Alvarez lens for high-speed focusing.

We present a modified configuration of a tunable Alvarez lens with a refocusing frequency of 1 kHz or more. In contrast to the classic Alvarez lens, the approach does not utilize a translational motion of two sub-lenses with respect to each other, but uses a 4f-setup to image two diffractive sub-lenses onto each other. Hereby focus tuning is achieved by rotating a galvo-mirror which affects the overlap of the two sub-lenses which together form an effective lens of refractive power which depends on the rotation angle of the galvo-mirror. We have demonstrated tuning of the optical power in a system where the diffractive Alvarez lens is realized by an LCOS-SLM. We consider our Alvarez setup especially suitable for applications where high refocusing rates are important, as for example in 3D life cell monitoring or tracking.

Vortex-free phase profiles for uniform patterning with computer-generated holography.

Computer-generated holography enables efficient light pattern generation through phase-only wavefront modulation. While perfect patterning usually requires control over both phase and amplitude, iterative Fourier transform algorithms (IFTA) can achieve phase-only approximations which maximize light efficiency at the cost of uniformity. The phase being unconstrained in the output plane, it can vary abruptly in some regions leading to destructive interferences. Among such structures phase vortices are the most common. Here we demonstrate theoretically, numerically and experimentally, a novel approach for eliminating phase vortices by spatially filtering the phase input to the IFTA, combining it with phase-based complex amplitude control at the spatial light modulator (SLM) plane to generate smooth shapes. The experimental implementation is achieved performing complex amplitude modulation with a phase-only SLM. This proposed experimental scheme offers a continuous and centered field of excitation. Lastly, we characterize achievable trade-offs between pattern uniformity, diffraction efficiency, and axial confinement.

Colored point spread function engineering for parallel confocal microscopy.

Using the color selectivity of a spatial light modulator (SLM) for both, tailoring the excitation beam at one wavelength, and multiplexing the image at the red-shifted fluorescence wavelength, it is possible to parallelize confocal microscopy, i.e. to simultaneously detect an axial stack (z-stack) of a sample. For this purpose, two diffractive patterns, one steering the excitation light, and the other manipulating the emission light, are combined within the same area of the SLM, which acts as a pure phase modulator. A recently demonstrated technique allows one to combine the patterns with high diffraction efficiency and low crosstalk, using the extended phase shifting capability of the SLM, which covers multiples of 2π at the respective wavelengths. For a first demonstration we compare standard confocal imaging with simultaneous image acquisition in two separate sample planes, which shows comparable results.

High-resolution confocal Raman microscopy using pixel reassignment.

We present a practical modification of fiber-coupled confocal Raman scanning microscopes that is able to provide high confocal resolution in conjunction with high light collection efficiency. For this purpose, the single detection fiber is replaced by a hexagonal lenslet array in combination with a hexagonally packed round-to-linear multimode fiber bundle. A multiline detector is used to collect individual Raman spectra for each fiber. Data post-processing based on pixel reassignment allows one to improve the lateral resolution by up to 41% compared to a single fiber of equal light collection efficiency. We present results from an experimental implementation featuring seven collection fibers, yielding a resolution improvement of about 30%. We believe that our implementation represents an attractive upgrade for existing confocal Raman microscopes that employ multi-line detectors.

Tilt-effect of holograms and images displayed on a spatial light modulator.

We show that a liquid crystal spatial light modulator (LCOS-SLM) can be used to display amplitude images, or phase holograms, which change in a pre-determined way when the display is tilted, i.e. observed under different angles. This is similar to the tilt-effect (also called "latent image effect") known from various security elements ("kinegrams") on credit cards or bank notes. The effect is achieved without any specialized optical components, simply by using the large phase shifting capability of a "thick" SLM, which extends over several multiples of 2π, in combination with the angular dependence of the phase shift. For hologram projection one can use the fact that the phase of a monochromatic wave is only defined modulo 2π. Thus one can design a phase pattern extending over several multiples of 2π, which transforms at different readout angles into different 2π-wrapped phase structures, due to the angular dependence of the modulo 2π operation. These different beams then project different holograms at the respective readout angles. In amplitude modulation mode (with inserted polarizer) the intensity of each SLM pixel oscillates over several periods when tuning its control voltage. Since the oscillation period depends on the readout angle, it is possible to find a certain control voltage which produces two (or more) selectable gray levels at a corresponding number of pre-determined readout angles. This is done with all SLM pixels individually, thus constructing different images for the selected angles. We experimentally demonstrate the reconstruction of multiple (Fourier- and Fresnel-) holograms, and of different amplitude images, by readout of static diffractive patterns in a variable angular range between 0° and 60°.

Direct measurement of axial optical forces.

Direct measurement of optical forces based on recording the change of momentum between the in- and outgoing light does not have specific requirements on particle size or shape, or on beam shape. Thus this approach overcomes many of the limitations of force measurements based on position measurements, which require frequent calibration. In this work we validate the achievable accuracy for direct force measurements in the axial direction for a single beam optical tweezers setup, based on numerical simulations and experimental investigations of situations, where the true force is known. We find that for typical experimental situations a good accuracy with an error of less than 1 % of the maximum force can be achieved, independent of particle size or refractive index, provided that the total amount of light scattered in the backward direction is also taken into account, which is easy to accomplish experimentally. Due to the inherent particle shape independence of the direct force measurement method, these findings support that it provides accurate results for 3D force measurements for particles of arbitrary shape.

Adjustable diffractive spiral phase plates.

We report on the fabrication and the experimental demonstration of Moiré diffractive spiral phase plates with adjustable helical charge. The proposed optical unit consists of two axially stacked diffractive elements of conjugate structure. The joint transmission function of the compound system corresponds to that of a spiral phase plate where the angle of mutual rotation about the central axis enables continuous adjustment of the helical charge. The diffractive elements are fabricated by gray-scale photolithography with a pixel size of 200 nm and 128 phase step levels in fused silica. We experimentally demonstrate the conversion of a TEM(00) beam into approximated Laguerre-Gauss (LG) beams of variable helical charge, with a correspondingly variable radius of their ring-shaped intensity distribution.

How to use a phase-only spatial light modulator as a color display.

We demonstrate that a parallel aligned liquid crystal on silicon (PA-LCOS) spatial light modulator (SLM) without any attached color mask can be used as a full color display with white light illumination. The method is based on the wavelength dependence of the (voltage controlled) birefringence of the liquid crystal pixels. Modern SLMs offer a wide range over which the birefringence can be modulated, leading (in combination with a linear polarizer) to several intensity modulation periods of a reflected light wave as a function of the applied voltage. Because of dispersion, the oscillation period strongly depends on the wavelength. Thus each voltage applied to an SLM pixel corresponds to another reflected color spectrum. For SLMs with a sufficiently broad tuning range, one obtains a color palette (i.e., a "color lookup-table"), which allows one to display color images. An advantage over standard liquid crystal displays (LCDs), which use color masks in front of the individual pixels, is that the light efficiency and the display resolution are increased by a factor of three.

Colour hologram projection with an SLM by exploiting its full phase modulation range.

We demonstrate independent and simultaneous manipulation of light beams of different wavelengths by a single hologram, which is displayed on a phase-only liquid crystal spatial light modulator (SLM). The method uses the high dynamic phase modulation range of modern SLMs, which can shift the phase of each pixel in a range between 0 up to 10π, depending on the readout wavelength. The extended phase range offers additional degrees of freedom for hologram encoding. Knowing the phase modulation properties of the SLM (i.e. the so-called lookup table) in the entire exploited wavelength range, an exhaustive search algorithm allows to combine different independently calculated 2π-holograms into a multi-level hologram with a phase range extending over several multiples of 2π. The combined multi-level hologram then reconstructs the original diffractive patterns with only small phase errors at preselected wavelengths, thus projecting the desired image fields almost without any crosstalk. We demonstrate this feature by displaying a static hologram at an SLM which is read out with an incoherent red-green-blue (RGB) beam, projecting a color image at a camera chip. This is done for both, a Fourier setup which needs a lens for image focusing, and in a "lensless" Fresnel setup, which also avoids the appearance of a focused zero-order spot in the image center. The experimentally obtained efficiency of a two-colour combination is on the order of 83% for each wavelength, with a crosstalk level between the two colour channels below 2%, whereas a three-colour combination still reaches an efficiency of about 60% and a crosstalk level below 5%.

Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation.

We present an approach for point spread function (PSF) engineering that allows one to shape the optical wavefront independently in both polarisation directions, with two adjacent phase masks displayed on a single liquid-crystal spatial light modulator (LC-SLM). The set-up employs a polarising beam splitter and a geometric image rotator to rectify and process both polarisation directions detected by the camera. We shape a single-lobe ("corkscrew") PSF that rotates upon defocus for each polarisation channel and combine the two polarisation channels with a relative 180° phase-shift on the computer, merging them into a single PSF that exhibits two lobes whose orientation contains information about the axial position. A major advantage lies in the possibility to measure and eliminate the aberrations in the two polarisation channels independently. We demonstrate axial super-localisation of isotropically emitting fluorescent nanoparticles. Our implementation of the single-lobe PSFs follows the method proposed by Prasad [Opt. Lett.38, 585 (2013)], and thus is to the best of our knowledge the first experimental realisation of this suggestion. For comparison we also study an approach with a rotating double-helix PSFs (in only one polarisation channel) and ascertain the trade-off between localisation precision and axial working range.

Broadband suppression of the zero diffraction order of an SLM using its extended phase modulation range.

The diffraction efficiency of a hologram displayed on a phase-only spatial light modulator (SLM) is maximal, if the SLM modulates the phase of the diffracted beam in a range between 0 and 2π. However, if the readout wavelength changes, or a broadband beam is used, due to dispersion this ideal modulation range cannot be maintained, which leads to lower diffraction efficiency and to the appearance of an undesired intense zero diffraction order. Here we show how an SLM with an extended phase modulation range of 4π can be used to display on-axis holograms with a strong suppression of the zero diffraction order in a wide spectral range, extending over 200 nm. The basic idea is to transform the original on-axis hologram into an off-axis hologram by adding a blazed grating and performing a modulo 2π operation, and then transforming it back by adding the conjugate grating, but without performing a subsequent modulo operation. The final hologram then spans over a phase range of 4π. The total diffracted field corresponds to that of the original on-axis hologram, but now the zero-order Fourier component is diffracted away from the optical axis. The same principle can be used to entangle the on-axis hologram with other phase structures, e.g. a random phase mask or a second hologram structure, followed by a subsequent addition of the conjugate mask, which may also suppress higher diffraction orders. The reconstructed holograms show a strong contrast enhancement in a broad wavelength range.

Lensless imaging through thin diffusive media.

Objects imaged through thin scattering media can be reconstructed with the knowledge of the complex transmission function of the diffuser. We demonstrate image reconstruction of static and dynamic objects with numerical phase conjugation in a lensless setup. Data is acquired by single shot intensity capture of an object coherently illuminated and obscured by an inhomogeneous medium, i.e. light diffracted at a specimen is scattered by a polycarbonate diffuser and the resulting speckle field is recorded. As a preparational step, which has to be performed only one time before imaging, the complex speckle field diffracted by the diffuser to the camera chip is measured interferometrically, which allows to reconstruct the transmission function of the diffuser. After insertion of the specimen, the speckle field in the camera plane changes, and the complex field of the sample can be reconstructed from the new intensity distribution. After initial interferometric measurement of the diffuser field, the method is robust with respect to a subsequent misalignment of the diffuser. The method can be extended to image objects placed between a pair of thin scattering plates. Since the object information is contained in a single speckle intensity pattern, it is possible to image dynamic processes at video rate.