Universal adaptive optics for microscopy through embedded neural network control.

The resolution and contrast of microscope imaging is often affected by aberrations introduced by imperfect optical systems and inhomogeneous refractive structures in specimens. Adaptive optics (AO) compensates these aberrations and restores diffraction limited performance. A wide range of AO solutions have been introduced, often tailored to a specific microscope type or application. Until now, a universal AO solution - one that can be readily transferred between microscope modalities - has not been deployed. We propose versatile and fast aberration correction using a physics-based machine learning assisted wavefront-sensorless AO control (MLAO) method. Unlike previous ML methods, we used a specially constructed neural network (NN) architecture, designed using physical understanding of the general microscope image formation, that was embedded in the control loop of different microscope systems. The approach means that not only is the resulting NN orders of magnitude simpler than previous NN methods, but the concept is translatable across microscope modalities. We demonstrated the method on a two-photon, a three-photon and a widefield three-dimensional (3D) structured illumination microscope. Results showed that the method outperformed commonly-used modal-based sensorless AO methods. We also showed that our ML-based method was robust in a range of challenging imaging conditions, such as 3D sample structures, specimen motion, low signal to noise ratio and activity-induced fluorescence fluctuations. Moreover, as the bespoke architecture encapsulated physical understanding of the imaging process, the internal NN configuration was no-longer a "black box", but provided physical insights on internal workings, which could influence future designs.

Laser-Written Tunable Liquid Crystal Aberration Correctors.

In this Article, we present a series of novel laser-written liquid crystal (LC) devices for aberration control for applications in beam shaping or aberration correction through adaptive optics. Each transparent LC device can correct for a chosen aberration mode with continuous greyscale tuning up to a total magnitude of more than 2π radians phase difference peak to peak at a wavelength of λ = 660 nm. For the purpose of demonstration, we present five different devices for the correction of five independent Zernike polynomial modes (although the technique could readily be used to manufacture devices based on other modes). Each device is operated by a single electrode pair tuned between 0 and 10 V. These devices have potential as a low-cost alternative to spatial light modulators for applications where a low-order aberration correction is sufficient and transmissive geometries are required.

Zwitterion-doped liquid crystal speckle reducers for immersive displays and vectorial imaging.

Lasers possess many attractive features (e.g., high brightness, narrow linewidth, well-defined polarization) that make them the ideal illumination source for many different scientific and technological endeavors relating to imaging and the display of high-resolution information. However, their high-level of coherence can result in the formation of noise, referred to as speckle, that can corrupt and degrade images. Here, we demonstrate a new electro-optic technology for combatting laser speckle using a chiral nematic liquid crystal (LC) dispersed with zwitterionic dopants. Results are presented that demonstrate when driven at the optimum electric field conditions, the speckle noise can be reduced by >90% resulting in speckle contrast (C) values of C = 0.07, which is approaching that required to be imperceptible to the human eye. This LC technology is then showcased in an array of different display and imaging applications, including a demonstration of speckle reduction in modern vectorial laser-based imaging.

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.

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.

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.

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.

Repeated imaging through a multimode optical fiber using adaptive optics.

Multimode optical fibers (MMF) have shown considerable potential for minimally invasive diffraction-limited fluorescence imaging of deep brain regions owing to their small size. They also look to be suitable for imaging across long time periods, with repeated measurements performed within the same brain region, which is useful to assess the role of synapses in normal brain function and neurological disease. However, the approach is not without challenge. Prior to imaging, light propagation through a MMF must be characterized in a calibration procedure. Manual repositioning, as required for repeated imaging, renders this calibration invalid. In this study, we provide a two-step solution to the problem consisting of (1) a custom headplate enabling precise reinsertion of the MMF implant achieving low-quality focusing and (2) sensorless adaptive optics to correct translational shifts in the MMF position enabling generation of high-quality imaging foci. We show that this approach achieves fluorescence imaging after repeated removal and reinsertion of a MMF.

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.

Laser induced forward transfer isolating complex-shaped cell by beam shaping.

Beam shaping techniques have been widely used in holographic optical tweezers to accurately manipulate tiny particles and hologram optimization algorithms have also been widely reported to improve the optical trapping performance. In this paper, we presented a beam shaping laser induced forward transfer (BS-LIFT) technique to isolate complex-shaped cells. To do this, we built up a BS-LIFT instrument which combined beam shaping methods and laser induced forward transfer using liquid-crystal-on-silicon spatial light modulator. The laser beam was modulated into multiple desired points at the focal plane employing the Gerchberg-Saxton (GS) algorithm. Feasibility was verified through transferring various samples. To our knowledge, this is the first demonstration of BS-LIFT applied to the transfer complex-shaped cells. We successfully transferred cells whose size ranged from 1 µm to 100 µm. Our design will provide a novel approach for the application of this beam shaping technique and the isolation of single cells with variable shapes.

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.

Shrinking multiplexed orbital angular momentum to the nanoscale.

Orbital angular momentum interactions at the nanoscale have remained elusive because the phase structure becomes unresolved. Now researchers have shown how to overcome this with tightly focused beams, demonstrating a record-high six-dimensional encoding in an ultra-dense nanoscale volume.

Polarisation optics for biomedical and clinical applications: a review.

Many polarisation techniques have been harnessed for decades in biological and clinical research, each based upon measurement of the vectorial properties of light or the vectorial transformations imposed on light by objects. Various advanced vector measurement/sensing techniques, physical interpretation methods, and approaches to analyse biomedically relevant information have been developed and harnessed. In this review, we focus mainly on summarising methodologies and applications related to tissue polarimetry, with an emphasis on the adoption of the Stokes-Mueller formalism. Several recent breakthroughs, development trends, and potential multimodal uses in conjunction with other techniques are also presented. The primary goal of the review is to give the reader a general overview in the use of vectorial information that can be obtained by polarisation optics for applications in biomedical and clinical research.

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.

3D super-resolution deep-tissue imaging in living mice.

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

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.

Democratising "Microscopi": a 3D printed automated XYZT fluorescence imaging system for teaching, outreach and fieldwork.

Commercial fluorescence microscope stands and fully automated XYZt fluorescence imaging systems are generally beyond the limited budgets available for teaching and outreach. We have addressed this problem by developing "Microscopi", an accessible, affordable, DIY automated imaging system that is built from 3D printed and commodity off-the-shelf hardware, including electro-mechanical, computer and optical components. Our design features automated sample navigation and image capture with a simple web-based graphical user interface, accessible with a tablet or other mobile device. The light path can easily be switched between different imaging modalities. The open source Python-based control software allows the hardware to be driven as an integrated imaging system. Furthermore, the microscope is fully customisable, which also enhances its value as a learning tool. Here, we describe the basic design and demonstrate imaging performance for a range of easily sourced specimens.