Ultrasound-induced reorientation for multi-angle optical coherence tomography.

Organoid and spheroid technology provide valuable insights into developmental biology and oncology. Optical coherence tomography (OCT) is a label-free technique that has emerged as an excellent tool for monitoring the structure and function of these samples. However, mature organoids are often too opaque for OCT. Access to multi-angle views is highly desirable to overcome this limitation, preferably with non-contact sample handling. To fulfil these requirements, we present an ultrasound-induced reorientation method for multi-angle-OCT, which employs a 3D-printed acoustic trap inserted into an OCT imaging system, to levitate and reorient zebrafish larvae and tumor spheroids in a controlled and reproducible manner. A model-based algorithm was developed for the physically consistent fusion of multi-angle data from a priori unknown angles. We demonstrate enhanced penetration depth in the joint 3D-recovery of reflectivity, attenuation, refractive index, and position registration for zebrafish larvae, creating an enabling tool for future applications in volumetric imaging.

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.

Optofluidic adaptive optics in multi-photon microscopy.

Adaptive optics, in combination with multi-photon techniques, is a powerful approach to image deep into a specimen. Remarkably, virtually all adaptive optics schemes today rely on wavefront modulators that are reflective, diffractive or both. This, however, can pose a severe limitation for applications. Here, we present a fast and robust sensorless adaptive optics scheme adapted for transmissive wavefront modulators. We study our scheme in numerical simulations and in experiments with a novel, optofluidic wavefront shaping device that is transmissive, refractive, polarisation-independent, and broadband. We demonstrate scatter correction of two-photon-excited fluorescence images of microbeads as well as brain cells and benchmark our device against a liquid-crystal spatial light modulator. Our method and technology could open new routes for adaptive optics in scenarios where previously, the restriction to reflective and diffractive devices may have staggered innovation and progress.

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.

Fast holographic scattering compensation for deep tissue biological imaging.

Scattering in biological tissues is a major barrier for in vivo optical imaging of all but the most superficial structures. Progress toward overcoming the distortions caused by scattering in turbid media has been made by shaping the excitation wavefront to redirect power into a single point in the imaging plane. However, fast, non-invasive determination of the required wavefront compensation remains challenging. Here, we introduce a quickly converging algorithm for non-invasive scattering compensation, termed DASH, in which holographic phase stepping interferometry enables new phase information to be updated after each measurement. This leads to rapid improvement of the wavefront correction, forming a focus after just one measurement iteration and achieving an order of magnitude higher signal enhancement at this stage than the previous state-of-the-art. Using DASH, we demonstrate two-photon fluorescence imaging of microglia cells in highly turbid mouse hippocampal tissue down to a depth of 530 µm.

Controlled orientation and sustained rotation of biological samples in a sono-optical microfluidic device.

In cell biology, recently developed technologies for studying suspended cell clusters, such as organoids or cancer spheroids, hold great promise relative to traditional 2D cell cultures. There is, however, growing awareness that sample confinement, such as fixation on a surface or embedding in a gel, has substantial impact on cell clusters. This creates a need for contact-less tools for 3D manipulation and inspection. This work addresses this demand by presenting a reconfigurable, hybrid sono-optical system for contact-free 3D manipulation and imaging, which is suitable for biological samples up to a few hundreds of micrometers in liquid suspension. In our sono-optical device, three independently addressable MHz transducers, an optically transparent top-transducer for levitation and two side-transducers, provide ultrasound excitation from three orthogonal directions. Steerable holographic optical tweezers give us an additional means of manipulation of the acoustically trapped specimen with high spatial resolution. We demonstrate how to control the reorientation or the spinning of complex samples, for instance for 3D visual inspection or for volumetric reconstruction. Whether continuous rotation or transient reorientation takes place depends on the strength of the acoustic radiation torque, arising from pressure gradients, compared to the acoustic viscous torque, arising from the shear forces at the viscous boundary layer around the particle. Based on numerical simulations and experimental insights, we develop a strategy to achieve a desired alignment or continuous rotation around a chosen axis, by tuning the relative strengths of the transducers and thus adjusting the relative contributions of viscous and radiation torques. The approach is widely applicable, as we discuss in several generic examples, with limitations dictated by size and shape asymmetry of the samples.

Simultaneous scattering compensation at multiple points in multi-photon microscopy.

The two-photon fluorescence imaging depth has been significantly improved in recent years by compensating for tissue scattering with wavefront correction. However, in most approaches the wavefront corrections are valid only over a small sample region on the order of 1 to 10 µm. In samples where most scattering structures are confined to a single plane, sample conjugate correction geometries can increase the observable field to a few tens of µm. Here, we apply a recently introduced fast converging scheme for sensor-less scattering correction termed "Dynamic Adaptive Scattering compensation Holography" (DASH) in a sample conjugate configuration with a high pixel count nematic liquid crystal spatial light modulator (LC-SLM). Using a large SLM allows us to simultaneously correct for scattering at multiple field points, which can be distributed over the entire field of view provided by the objective lens. Despite the comparably slow refresh time of LC-SLMs, we achieve correction times on the order of 10 s per field point, which we show is sufficiently fast to counteract scattering at multiple sites in living mouse hippocampal tissue slices.

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.

High-NA two-photon single cell imaging with remote focusing using a diffractive tunable lens.

Fast, volumetric structural and functional imaging of cellular and sub-cellular dynamics inside the living brain is one of the most desired capabilities in the neurosciences, but still faces serious challenges. Specifically, while few solutions for rapid 3D scanning exist, it is generally much easier to facilitate fast in-plane scanning than it is to scan axially at high speeds. Remote focusing in which the imaging plane is shifted along the optical axis by a tunable lens while maintaining the position of the sample and objective is a promising approach to increase the axial scan speed, but existing techniques often introduce severe optical aberrations in high-NA imaging systems, eliminating the possibility of diffraction-limited single-cell imaging. Here, we demonstrate near diffraction-limited, volumetric two-photon fluorescence microscopy in which we resolve the deep sub-micron structures of single microglia cells with axial scanning performed using a novel high-NA remote focusing method. Image contrast is maintained to within 7% compared to mechanical sample stepping and the focal volume remains nearly diffraction-limited over an axial range greater than 86 µm.

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.

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.

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.

Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains.

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm-1 to 2800 cm-1. Raman spectra illustrated a similar picture with less ν2PO43-at 450 cm-1 and ν4PO43- from 590 cm-1 to 584 cm-1, amide III at 1272 cm-1 and protein CH2 deformation at 1446 cm-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains.

Orbital angular momentum light in microscopy.

Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application.This article is part of the themed issue 'Optical orbital angular momentum'.

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.

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.

Acoustic force mapping in a hybrid acoustic-optical micromanipulation device supporting high resolution optical imaging.

Many applications in the life-sciences demand non-contact manipulation tools for forceful but nevertheless delicate handling of various types of sample. Moreover, the system should support high-resolution optical imaging. Here we present a hybrid acoustic/optical manipulation system which utilizes a transparent transducer, making it compatible with high-NA imaging in a microfluidic environment. The powerful acoustic trapping within a layered resonator, which is suitable for highly parallel particle handling, is complemented by the flexibility and selectivity of holographic optical tweezers, with the specimens being under high quality optical monitoring at all times. The dual acoustic/optical nature of the system lends itself to optically measure the exact acoustic force map, by means of direct force measurements on an optically trapped particle. For applications with (ultra-)high demand on the precision of the force measurements, the position of the objective used for the high-NA imaging may have significant influence on the acoustic force map in the probe chamber. We have characterized this influence experimentally and the findings were confirmed by model simulations. We show that it is possible to design the chamber and to choose the operating point in such a way as to avoid perturbations due to the objective lens. Moreover, we found that measuring the electrical impedance of the transducer provides an easy indicator for the acoustic resonances.

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°.