Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

Python-Microscope - a new open-source Python library for the control of microscopes.

Custom-built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and that is translatable between components from different manufacturers. Here we report Python-Microscope, a free and open-source Python library for high-performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware, and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architectural features of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: integration with user-facing projects, namely the Microscope-Cockpit project; control of complex microscopes at high speed while using the Python programming language; and use as a microscope simulation tool for software development.

Deflectometry based calibration of a deformable mirror for aberration correction and remote focusing in microscopy.

Adaptive optics (AO) techniques enhance the capability of optical microscopy through precise control of wavefront modulations to compensate phase aberrations and improves image quality. However, the aberration correction is often limited due to the lack of dynamic range in existing calibration methods, such as interferometry or Shack-Hartmann (SH) wavefront sensors. Here, we use deflectometry (DF) as a calibration method for a deformable mirror (DM) to extend the available range of aberration correction. We characterised the dynamic range and accuracy of the DF-based calibration of DMs depending on the spatial frequency of the test pattern used in DF. We also demonstrated the capability of large magnitude phase control for remote-focusing over a range larger than was possible with SH sensing.

Generalised adaptive optics method for high-NA aberration-free refocusing in refractive-index-mismatched media.

Phase aberrations are introduced when focusing by a high-numerical aperture (NA) objective lens into refractive-index-mismatched (RIM) media. The axial focus position in these media can be adjusted through either optical remote-focusing or mechanical stage translation. Despite the wide interest in remote-focusing, no generalised control algorithm using Zernike polynomials has been presented that performs independent remote-focusing and RIM correction in combination with mechanical stage translation. In this work, we thoroughly review derivations that model high-NA defocus and RIM aberration. We show through both numerical simulation and experimental results that optical remote-focusing using an adaptive device and mechanical stage translation are not optically equivalent processes, such that one cannot fully compensate for the other without additional aberration compensation. We further establish new orthogonal modes formulated using conventional Zernike modes and discuss its device programming to control high-NA remote-focusing and RIM correction as independent primary modes in combination with mechanical stage translation for aberration-free refocusing. Numerical simulations are performed, and control algorithms are validated experimentally by fabricating graphitic features in diamond using direct laser writing.

Single-mode sapphire fiber Bragg grating.

Sapphire optical fiber has the ability to withstand ultrahigh temperatures and high radiation, but it is multimoded which prevents its use in many sensing applications. Problematically, Bragg gratings in such fiber exhibit multiple reflection peaks with a fluctuating power distribution. In this work, we write single-mode waveguides with Bragg gratings in sapphire using a novel multi-layer depressed cladding design in the 1550 nm telecommunications waveband. The Bragg gratings have a narrow bandwidth (<0.5 nm) and have survived annealing at 1000°C. The structures are inscribed with femtosecond laser direct writing, using adaptive beam shaping with a non-immersion objective. A single-mode sapphire fiber Bragg grating is created by writing a waveguide with a Bragg grating within a 425 µm diameter sapphire optical fiber, providing significant potential for accurate remote sensing in ultra-extreme environments.

Dynamic phase measurement of fast liquid crystal phase modulators.

We present dynamic time-resolved measurements of a multi-pixel analog liquid crystal phase modulator driven at a 1 kHz frame rate. A heterodyne interferometer is used to interrogate two pixels independently and simultaneously, to deconvolve phase modulation with a wide bandwidth. The root mean squared optical phase error within a 30 Hz to 25 kHz bandwidth is <0.5° and the crosstalk rejection is 50 dB. Measurements are shown for a custom-built device with a flexoelectro-optic chiral nematic liquid crystal. However, the technique is applicable to many different types of optical phase modulators and spatial light modulators.

Wavefront-sensorless adaptive optics with a laser-free spinning disk confocal microscope.

Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution.

Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning.

Rapid autofocusing over long distances is critical for tracking 3D topological variations and sample motion in real time. Taking advantage of a deformable mirror and Shack-Hartmann wavefront sensor, remote focusing can permit fast axial scanning with simultaneous correction of system-induced aberrations. Here, we report an autofocusing technique that combines remote focusing with sequence-dependent learning via a bidirectional long short term memory network. A 120 µm autofocusing range was achieved in a compact reflectance confocal microscope both in air and in refractive-index-mismatched media, with similar performance under arbitrary-thickness liquid layers up to 1 mm. The technique was validated on sample types not used for network training, as well as for tracking of continuous axial motion. These results demonstrate that the proposed technique is suitable for real-time aberration-free autofocusing over a large axial range, and provides unique advantages for biomedical, holographic and other related applications.

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.

z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics.

Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.

Trimming laser-written waveguides through overwriting.

Femtosecond laser direct writing is widely used to create waveguide circuits for optical processing in applications including communications, astrophotonics, simulation and quantum information processing. The properties of these waveguide circuits can be sensitive to the fabrication conditions, meaning that noticeable variability can be present in nominally identical manufactured components. One potential solution to this problem is the use of device trimming, whereby additional laser fabrication is applied to optimise the optical properties of a device based upon measurement feedback. We show how this approach can be used in the manufacture of directional couplers by overwriting the laser-written structure to alter the coupling ratios.

Microscope-AOtools: a generalised adaptive optics implementation.

Aberrations arising from sources such as sample heterogeneity and refractive index mismatches are constant problems in biological imaging. These aberrations reduce image quality and the achievable depth of imaging, particularly in super-resolution microscopy techniques. Adaptive optics (AO) technology has been proven to be effective in correcting for these aberrations, thereby improving the image quality. However, it has not been widely adopted by the biological imaging community due, in part, to difficulty in set-up and operation of AO. The methods for doing so are not novel or unknown, but new users often waste time and effort reimplementing existing methods for their specific set-ups, hardware, sample types, etc. Microscope-AOtools offers a robust, easy-to-use implementation of the essential methods for set-up and use of AO elements and techniques. These methods are constructed in a generalised manner that can utilise a range of adaptive optics elements, wavefront sensing techniques and sensorless AO correction methods. Furthermore, the methods are designed to be easily extensible as new techniques arise, leading to a streamlined pipeline for new AO technology and techniques to be adopted by the wider microscopy community.

Multi-scale sensorless adaptive optics: application to stimulated emission depletion microscopy.

Sensorless adaptive optics is commonly used to compensate specimen-induced aberrations in high-resolution fluorescence microscopy, but requires a bespoke approach to detect aberrations in different microscopy techniques, which hinders its widespread adoption. To overcome this limitation, we propose using wavelet analysis to quantify the loss of resolution due to the aberrations in microscope images. By examining the variations of the wavelet coefficients at different scales, we are able to establish a multi-valued image quality metric that can be successfully deployed in different microscopy techniques. To corroborate our arguments, we provide experimental verification of our method by performing aberration correction experiments in both confocal and STED microscopy using three different specimens.

Anisoplanatic adaptive optics in parallelized laser scanning microscopy.

Inhomogeneities in the refractive index of a biological microscopy sample can introduce phase aberrations, severely impairing the quality of images. Adaptive optics can be employed to correct for phase aberrations and improve image quality. However, conventional adaptive optics can only correct a single phase aberration for the whole field of view (isoplanatic correction) while, due to the highly heterogeneous nature of biological tissues, the sample induced aberrations in microscopy often vary throughout the field of view (anisoplanatic aberration), limiting significantly the effectiveness of adaptive optics. This paper reports on a new approach for aberration correction in laser scanning confocal microscopy, in which a spatial light modulator is used to generate multiple excitation points in the sample to simultaneously scan different portions of the field of view with completely independent correction, achieving anisoplanatic compensation of sample induced aberrations, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions. The method was tested in whole Drosophila brains and in larval Zebrafish, each showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.

Practical sensorless aberration estimation for 3D microscopy with deep learning.

Estimation of optical aberrations from volumetric intensity images is a key step in sensorless adaptive optics for 3D microscopy. Recent approaches based on deep learning promise accurate results at fast processing speeds. However, collecting ground truth microscopy data for training the network is typically very difficult or even impossible thereby limiting this approach in practice. Here, we demonstrate that neural networks trained only on simulated data yield accurate predictions for real experimental images. We validate our approach on simulated and experimental datasets acquired with two different microscopy modalities and also compare the results to non-learned methods. Additionally, we study the predictability of individual aberrations with respect to their data requirements and find that the symmetry of the wavefront plays a crucial role. Finally, we make our implementation freely available as open source software in Python.

Volumetric two-photon fluorescence imaging of live neurons using a multimode optical fiber.

Multimode optical fibers (MMFs), combined with wavefront control methods, have achieved minimally invasive in vivo imaging of neurons in deep-brain regions with diffraction-limited spatial resolution. Here, we report a method for volumetric two-photon fluorescence imaging with a MMF-based system requiring a single transmission matrix measurement. Central to this method is the use of a laser source able to generate both continuous wave light and femtosecond pulses. The chromatic dispersion of pulses generated an axially elongated excitation focus, which we used to demonstrate volumetric imaging of neurons and their dendrites in live rat brain slices through a 60 µm core MMF.

Photonic Topological Mode Bound to a Vortex.

We report the observation of a mode associated with a topological defect in the bulk of a 2D photonic material by introducing a vortex distortion to a hexagonal lattice analogous to graphene. The observed modes lie midgap at zero energy and are closely related to Majorana bound states in superconducting vortices. This is the first experimental demonstration of the Jackiw-Rossi model [R. Jackiw and P. Rossi, Nucl. Phys. B190, 681 (1981)NUPBBO0550-321310.1016/0550-3213(81)90044-4].

Laser Writing of Scalable Single Color Centers in Silicon Carbide.

Single photon emitters in silicon carbide (SiC) are attracting attention as quantum photonic systems ( Awschalom et al. Nat. Photonics 2018 , 12 , 516 - 527 ; Atatüre et al. Nat. Rev. Mater. 2018 , 3 , 38 - 51 ). However, to achieve scalable devices, it is essential to generate single photon emitters at desired locations on demand. Here we report the controlled creation of single silicon vacancy (VSi) centers in 4H-SiC using laser writing without any postannealing process. Due to the aberration correction in the writing apparatus and the nonannealing process, we generate single VSi centers with yields up to 30%, located within about 80 nm of the desired position in the transverse plane. We also investigated the photophysics of the laser writing VSi centers and concluded that there are about 16 photons involved in the laser writing VSi center process. Our results represent a powerful tool in the fabrication of single VSi centers in SiC for quantum technologies and provide further insights into laser writing defects in dielectric materials.

Calibration of a phase-only spatial light modulator for both phase and retardance modulation.

Liquid crystal spatial light modulators (SLMs) are usually configured and calibrated for phase modulation. However, as they are variable retarders, they also have application as polarization modulators. We show that conventional phase-only calibrations are insufficient for this purpose, and a separate retardance calibration is needed. To overcome this shortcoming we report a simple Twyman-Green interferometer-based setup to realize SLM phase and retardance calibration. For phase calibration, we identify the non-linear, spatially variant response to the drive voltage of the SLM using fringe analysis and both horizontally and vertically polarized incident light. For retardance calibration, we use incident light polarized at 45° and assess the intensity variation. The methods presented are compatible with in situ calibration of SLMs.