Low cross-talk optical addressing of trapped-ion qubits using a novel integrated photonic chip.

Individual optical addressing in chains of trapped atomic ions requires the generation of many small, closely spaced beams with low cross-talk. Furthermore, implementing parallel operations necessitates phase, frequency, and amplitude control of each individual beam. Here, we present a scalable method for achieving all of these capabilities using a high-performance integrated photonic chip coupled to a network of optical fibre components. The chip design results in very low cross-talk between neighbouring channels even at the micrometre-scale spacing by implementing a very high refractive index contrast between the channel core and cladding. Furthermore, the photonic chip manufacturing procedure is highly flexible, allowing for the creation of devices with an arbitrary number of channels as well as non-uniform channel spacing at the chip output. We present the system used to integrate the chip within our ion trap apparatus and characterise the performance of the full individual addressing setup using a single trapped ion as a light-field sensor. Our measurements showed intensity cross-talk below ~10-3 across the chip, with minimum observed cross-talk as low as ~10-5.

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.

On-chip beam rotators, adiabatic mode converters, and waveplates through low-loss waveguides with variable cross-sections.

Photonics integrated circuitry would benefit considerably from the ability to arbitrarily control waveguide cross-sections with high precision and low loss, in order to provide more degrees of freedom in manipulating propagating light. Here, we report a new method for femtosecond laser writing of optical-fiber-compatible glass waveguides, namely spherical phase-induced multicore waveguide (SPIM-WG), which addresses this challenging task with three-dimensional on-chip light control. Fabricating in the heating regime with high scanning speed, precise deformation of cross-sections is still achievable along the waveguide, with shapes and sizes finely controllable of high resolution in both horizontal and vertical transversal directions. We observed that these waveguides have high refractive index contrast of 0.017, low propagation loss of 0.14 dB/cm, and very low coupling loss of 0.19 dB coupled from a single-mode fiber. SPIM-WG devices were easily fabricated that were able to perform on-chip beam rotation through varying angles, or manipulate the polarization state of propagating light for target wavelengths. We also demonstrated SPIM-WG mode converters that provide arbitrary adiabatic mode conversion with high efficiency between symmetric and asymmetric nonuniform modes; examples include circular, elliptical modes, and asymmetric modes from ppKTP (periodically poled potassium titanyl phosphate) waveguides which are generally applied in frequency conversion and quantum light sources. Created inside optical glass, these waveguides and devices have the capability to operate across ultra-broad bands from visible to infrared wavelengths. The compatibility with optical fiber also paves the way toward packaged photonic integrated circuitry, which usually needs input and output fiber connections.

Inverse design of gradient-index volume multimode converters.

Graded-index optical elements are capable of shaping light precisely and in very specific ways. While classical freeform optics uses only a two-dimensional domain such as the surface of a lens, recent technological advances in laser manufacturing offer promising prospects for the realization of arbitrary three-dimensional graded-index volumes, i.e. transparent dielectric substrates with voxel-wise modified refractive index distributions. Such elements would be able to perform complex light transformations on compact scales. Here we present an algorithmic approach for computing 3D graded-index devices, which utilizes numerical beam propagation and error reduction based on gradient descent. We present solutions for millimeter-sized elements addressing important tasks in photonics: a mode sorter, a photonic lantern and a multimode intensity beam shaper. We further discuss suitable cost functions for all designs to be used in the algorithm. The 3D graded-index designs are spatially smooth and require a relatively small refractive index range in the order of 10-2, which is within the reach of direct laser writing manufacturing processes such as two-photon polymerization.

Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy.

Single molecule localization microscopy (SMLM) has the potential to resolve structural details of biological samples at the nanometer length scale. Compared to room temperature experiments, SMLM performed under cryogenic temperature achieves higher photon yields and, hence, higher localization precision. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters with fixed orientation. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of 1 nm, thereby reaching the corresponding Cramér Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments.

Holographic beam shaping of partially coherent light.

We present an algorithmic approach for holographic shaping of partially coherent light, which is described by a mode expansion containing thousands of individual modes. Using gradient descent and algorithmic differentiation, our algorithm is able to find a set of axially separated phase patterns such that each mode undergoes an individually optimized transformation with respect to the formation of a user-defined target intensity distribution. We demonstrate numerically and experimentally that a tandem of two phase patterns can achieve any intensity profile transformation with good accuracy.

Tomographic refractive index profiling of direct laser written waveguides.

The fabrication of complex integrated photonic devices via direct laser writing is a powerful and rapidly developing technology. However, the approach is still facing several challenges. One of them is the reliable quantitative characterization of refractive index (RI) changes induced upon laser exposure. To this end, we develop a tomographic reconstruction algorithm following a modern optimization approach, relying on accelerated proximal gradient descent, based on intensity images only. Very recently, such algorithms have become the state of the art in the community of bioimaging, but have never been applied to direct laser written structures such as waveguides. We adapt the algorithm to our concern of characterizing these translation-invariant structures and extend it in order to jointly estimate the aberrations introduced by the imaging system. We show that a correct estimation of these aberrations is necessary to make use of data recorded at larger angles and that it can increase the fidelity of the reconstructed RI profiles. Moreover, we present a method allowing to cross-validate the RI reconstructions by comparing en-face widefield images of thin waveguide sections with matching simulations based on the retrieved RI profile.

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.

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.

Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence.

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

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.

Erratum: Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy: erratum.

[This corrects the article on p. 775 in vol. 11, PMID: 32206395.].

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.

Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy.

Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible.

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.

Spectral image scanning microscopy.

For decades, the confocal microscope has represented one of the dominant imaging systems in biomedical imaging at sub-cellular lengthscales. Recently, however, it has increasingly been replaced by a related, but more powerful successor technique termed image scanning microscopy (ISM). In this article, we present ISM capable of measuring spectroscopic information such as that contained in fluorescence or Raman images. Compared to established confocal spectroscopic imaging systems, our implementation offers similar spectral resolution, but higher spatial resolution and detection efficiency. Color sensitivity is achieved by a grating placed in the detection path in conjunction with a camera collecting both spatial and spectral information. The multidimensional data is processed using multi-view maximum likelihood image reconstruction. Our findings are supported by numerical simulations and experiments on micro beads and double-stained HeLa cells.

Two-photon PSF-engineered image scanning microscopy.

We present two-photon fluorescence image scanning microscopy (ISM) with engineered excitation and detection point-spread-functions enabling 3D imaging in a single 2D scan. This demonstration combines excitation using a holographic multispot array of focused femtosecond pulses with a high-efficiency single-helix PSF phase mask detection. Camera detection along with a multiview reconstruction algorithm allows volumetric imaging of biological samples over a depth of field spanning more than 1500 nm with an axial resolution of better than 400 nm. The nonlinear two-photon process improves sectioning and the inherent longer wavelengths increase the penetration depth in scattering samples. Our method extends the performance of 3D ISM towards thicker biological samples.

Four-dimensional light shaping: manipulating ultrafast spatiotemporal foci in space and time.

The spectral dispersion of ultrashort pulses allows the simultaneous focusing of light in both space and time, which creates so-called spatiotemporal foci. Such space-time coupling may be combined with the existing holographic techniques to give a further dimension of control when generating focal light fields. In the present study, it is shown that a phase-only hologram placed in the pupil plane of an objective and illuminated by a spatially chirped ultrashort pulse can be used to generate three-dimensional arrays of spatio-temporally focused spots. By exploiting the pulse front tilt generated at focus when applying simultaneous spatial and temporal focusing (SSTF), it is possible to overlap neighboring foci in time to create a smooth intensity distribution. The resulting light field displays a high level of axial confinement, with experimental demonstrations given through two-photon microscopy and the non-linear laser fabrication of glass.

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.