Generating Airy beams through multimode fibres.

Focussing light through a multimode fibre (MMF) is the basis of holographic endoscopes, which currently enable detailed imaging of deep tissue. Achieving high fidelity and purity diffraction-limited foci has been shown to be possible, when fully controlling the amplitude, phase, and two orthogonal polarisation states of the input field. Yet, generating more complex field distributions with similar performance remains to be assessed. Here, we demonstrate the generation of Airy beams through an MMF containing in excess of 90 % of the optical power delivered by the fibre. We discuss two distinct methods for generating optical landscapes: the direct field and the Fourier domain synthesis. Moreover, we showcase the flexibility of the Fourier domain synthesis to modify the generated beam.

Advancing the path to in-vivo imaging in freely moving mice via multimode-multicore fiber based holographic endoscopy.

Significance: Hair-thin multimode optical fiber-based holographic endoscopes have gained considerable interest in modern neuroscience for their ability to achieve cellular and even subcellular resolution during in-vivo deep brain imaging. However, the application of multimode fibers in freely moving animals presents a persistent challenge as it is difficult to maintain optimal imaging performance while the fiber undergoes deformations. Aim: We propose a fiber solution for challenging in-vivo applications with the capability of deep brain high spatial resolution imaging and neuronal activity monitoring in anesthetized as well as awake behaving mice. Approach: We used our previously developed M3CF multimode-multicore fiber to record fluorescently labeled neurons in anesthetized mice. Our M3CF exhibits a cascaded refractive index structure, enabling two distinct regimes of light transport that imitate either a multimode or a multicore fiber. The M3CF has been specifically designed for use in the initial phase of an in-vivo experiment, allowing for the navigation of the endoscope's distal end toward the targeted brain structure. The multicore regime enables the transfer of light to and from each individual neuron within the field of view. For chronic experiments in awake behaving mice, it is crucial to allow for disconnecting the fiber and the animal between experiments. Therefore, we provide here an effective solution and establish a protocol for reconnection of two segments of M3CF with hexagonally arranged corelets. Results: We successfully utilized the M3CF to image neurons in anaesthetized transgenic mice expressing enhanced green fluorescent protein. Additionally, we compared imaging results obtained with the M3CF with larger numerical aperture (NA) fibers in fixed whole-brain tissue. Conclusions: This study focuses on addressing challenges and providing insights into the use of multimode-multicore fibers as imaging solutions for in-vivo applications. We suggest that the upcoming version of the M3CF increases the overall NA between the two cladding layers to allow for access to high resolution spatial imaging. As the NA increases in the multimode regime, the fiber diameter and ring structure must be reduced to minimize the computational burden and invasiveness.

"There's plenty of room at the bottom": deep brain imaging with holographic endo-microscopy.

Significance: Over more than 300 years, microscopic imaging keeps providing fundamental insights into the mechanisms of living organisms. Seeing microscopic structures beyond the reach of free-space light-based microscopy, however, requires dissection of the tissue-an intervention seriously disturbing its physiological functions. The hunt for low-invasiveness tools has led a growing community of physicists and engineers into the realm of complex media photonics. One of its activities represents exploiting multimode optical fibers (MMFs) as ultra-thin endoscopic probes. Employing wavefront shaping, these tools only recently facilitated the first peeks at cells and their sub-cellular compartments at the bottom of the mouse brain with the impact of micro-scale tissue damage. Aim: Here, we aim to highlight advances in MMF-based holographic endo-microscopy facilitating microscopic imaging throughout the whole depth of the mouse brain. Approach: We summarize the important technical and methodological prerequisites for stabile high-resolution imaging in vivo. Results: We showcase images of the microscopic building blocks of brain tissue, including neurons, neuronal processes, vessels, intracellular calcium signaling, and red blood cell velocity in individual vessels. Conclusions: This perspective article helps to understand the complexity behind the technology of holographic endo-microscopy, summarizes its recent advances and challenges, and stimulates the mind of the reader for further exploitation of this tool in the neuroscience research.

110 µm thin endo-microscope for deep-brain in vivo observations of neuronal connectivity, activity and blood flow dynamics.

Light-based in-vivo brain imaging relies on light transport over large distances of highly scattering tissues. Scattering gradually reduces imaging contrast and resolution, making it difficult to reach structures at greater depths even with the use of multiphoton techniques. To reach deeper, minimally invasive endo-microscopy techniques have been established. These most commonly exploit graded-index rod lenses and enable a variety of modalities in head-fixed and freely moving animals. A recently proposed alternative is the use of holographic control of light transport through multimode optical fibres promising much less traumatic application and superior imaging performance. We present a 110 µm thin laser-scanning endo-microscope based on this prospect, enabling in-vivo volumetric imaging throughout the whole depth of the mouse brain. The instrument is equipped with multi-wavelength detection and three-dimensional random access options, and it performs at lateral resolution below 1 µm. We showcase various modes of its application through the observations of fluorescently labelled neurones, their processes and blood vessels. Finally, we demonstrate how to exploit the instrument to monitor calcium signalling of neurones and to measure blood flow velocity in individual vessels at high speeds.

Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

Near perfect focusing through multimode fibres.

Holographic, multimode fibre (MMF) based endoscopes envision high-quality in-vivo imaging inside previously inaccessible structures of living organisms, amongst other perspective applications. Within these instruments, a digital micro-mirror device (DMD) is deployed in order to holographically synthesise light fields which, after traversing the multimode fibre, form foci at desired positions behind the distal fibre facet. When applied in various imaging modalities, the purity and sharpness of the achieved foci are determinant for the imaging performance. Here we present diffraction-limited foci, which contain in excess of 96% of optical power delivered by the fibre which, to the best of our knowledge, represents the highest value reported to date. Further, we quantitatively study the impact of various conditions of the experimental procedure including input polarisation settings, influence of ghost diffraction orders, light modulation regimes, bias of the calibration camera and the influence of noise.

Suppression of the non-linear background in a multimode fibre CARS endoscope.

Multimode fibres show great potential for use as miniature endoscopes for imaging deep in tissue with minimal damage. When used for coherent anti-Stokes Raman scattering (CARS) microscopy with femtosecond excitation sources, a high band-width probe is required to efficiently focus the broadband laser pulses at the sample plane. Although graded-index (GRIN) fibres have a large bandwidth, it is accompanied by a strong background signal from four-wave mixing and other non-linear processes occurring inside the fibre. We demonstrate that using a composite probe consisting of a GRIN fibre with a spliced on step-index fibre reduces the intensity of the non-linear background by more than one order of magnitude without significantly decreasing the focusing performance of the probe. Using this composite probe we acquire CARS images of biologically relevant tissue such as myelinated axons in the brain with good contrast.

Time-of-flight 3D imaging through multimode optical fibers.

Time-of-flight three-dimensional (3D) imaging has applications that range from industrial inspection to motion tracking. Depth is recovered by measuring the round-trip flight time of laser pulses, typically using collection optics of several centimeters in diameter. We demonstrate near­video-rate 3D imaging through multimode fibers with a total aperture of several hundred micrometers. We implement aberration correction using wavefront shaping synchronized with a pulsed source and scan the scene at ~23,000 points per second. We image moving objects several meters beyond the end of an ~40-centimeters-long fiber of 50-micrometer core diameter at frame rates of ~5 hertz. Our work grants far-field depth-resolving capabilities to ultrathin microendoscopes, which we expect to have applications to clinical and remote inspection scenarios.

Computational image enhancement of multimode fibre-based holographic endo-microscopy: harnessing the muddy modes.

In imaging geometries, which employ wavefront-shaping to control the light transport through a multi-mode optical fibre (MMF), this terminal hair-thin optical component acts as a minimally invasive objective lens, enabling high resolution laser-scanning fluorescence microscopy inside living tissues at depths hardly accessible by any other light-based technique. Even in the most advanced systems, the diffraction-limited foci scanning the object across the focal plane are contaminated by a stray optical signal carrying typically few tens of % of the total optical power. The stray illumination takes the shape of a randomised but reproducible speckle, and is unique for each position of the focus. We experimentally demonstrate that the performance of imaging a fluorescent object can be significantly improved, when resulting images are computationally post-processed, utilising records of intensities of all speckle-contaminated foci used in the imaging procedure. We present two algorithms based on a regularised iterative inversion and regularised direct pseudo-inversion respectively which lead to enhancement of the image contrast and resolution.

Side-view holographic endomicroscopy via a custom-terminated multimode fibre.

Microendoscopes based on optical fibres have recently come to the fore as promising candidates allowing in-vivo observations of otherwise inaccessible biological structures in animal models. Despite being still in its infancy, imaging can now be performed at the tip of a single multimode fibre, by relying on powerful holographic methods for light control. Fibre based endoscopy is commonly performed en face, resulting in possible damage of the specimen owing to the direct contact between the distal end of the probe and target. On this ground, we designed an all-fibre probe with an engineered termination that reduces compression and damage to the tissue under investigation upon probe insertion. The geometry of the termination brings the field of view to a plane parallel to the fibre's longitudinal direction, conveying the probe with off-axis imaging capabilities. We show that its focusing ability also benefits from a higher numerical aperture, resulting in imaging with increased spatial resolution. The effect of probe insertion was investigated inside a tissue phantom comprising fluorescent particles suspended in agarose gel, and a comparison was established between the novel side-view probe and the standard en face fibre probe. This new concept paves the way to significantly less invasive deep-tissue imaging.

Time-averaged image projection through a multimode fiber.

Many disciplines, ranging from lithography to opto-genetics, require high-fidelity image projection. However, not all optical systems can display all types of images with equal ease. Therefore, the image projection quality is dependent on the type of image. In some circumstances, this can lead to a catastrophic loss of intensity or image quality. For complex optical systems, it may not be known in advance which types of images pose a problem. Here we show a new method called Time-Averaged image Projection (TAP), allowing us to mitigate these limitations by taking the entire image projection system into account despite its complexity and building the desired intensity distribution up from multiple illumination patterns. Using a complex optical setup, consisting of a wavefront shaper and a multimode optical fiber illuminated by coherent light, we succeeded to suppress any speckle-related background. Further, we can display independent images at multiple distances simultaneously, and alter the effective sharpness depth through the algorithm. Our results demonstrate that TAP can significantly enhance the image projection quality in multiple ways. We anticipate that our results will greatly complement any application in which the response to light irradiation is relatively slow (one microsecond with current technology) and where high-fidelity spatial distribution of optical power is required.

Memory effect assisted imaging through multimode optical fibres.

When light propagates through opaque material, the spatial information it holds becomes scrambled, but not necessarily lost. Two classes of techniques have emerged to recover this information: methods relying on optical memory effects, and transmission matrix (TM) approaches. Here we develop a general framework describing the nature of memory effects in structures of arbitrary geometry. We show how this framework, when combined with wavefront shaping driven by feedback from a guide-star, enables estimation of the TM of any such system. This highlights that guide-star assisted imaging is possible regardless of the type of memory effect a scatterer exhibits. We apply this concept to multimode fibres (MMFs) and identify a 'quasi-radial' memory effect. This allows the TM of an MMF to be approximated from only one end - an important step for micro-endoscopy. Our work broadens the applications of memory effects to a range of novel imaging and optical communication scenarios.

All-optical manipulation of photonic membranes.

We demonstrate the all-optical manipulation of polymeric membranes in microfluidic environments. The membranes are decorated with handles for their use in holographic optical tweezers systems. Our results show that due to their form factor the membranes present a substantial increase in their mechanical stability, respect to micrometric dielectric particles. This intrinsic superior stability is expected to improve profoundly a wide range of bio-photonic applications that rely on the optical manipulation of micrometric objects.

Compressively sampling the optical transmission matrix of a multimode fibre.

The measurement of the optical transmission matrix (TM) of an opaque material is an advanced form of space-variant aberration correction. Beyond imaging, TM-based methods are emerging in a range of fields, including optical communications, micro-manipulation, and computing. In many cases, the TM is very sensitive to perturbations in the configuration of the scattering medium it represents. Therefore, applications often require an up-to-the-minute characterisation of the fragile TM, typically entailing hundreds to thousands of probe measurements. Here, we explore how these measurement requirements can be relaxed using the framework of compressive sensing, in which the incorporation of prior information enables accurate estimation from fewer measurements than the dimensionality of the TM we aim to reconstruct. Examples of such priors include knowledge of a memory effect linking the input and output fields, an approximate model of the optical system, or a recent but degraded TM measurement. We demonstrate this concept by reconstructing the full-size TM of a multimode fibre supporting 754 modes at compression ratios down to ∼5% with good fidelity. We show that in this case, imaging is still possible using TMs reconstructed at compression ratios down to ∼1% (eight probe measurements). This compressive TM sampling strategy is quite general and may be applied to a variety of other scattering samples, including diffusers, thin layers of tissue, fibre optics of any refractive profile, and reflections from opaque walls. These approaches offer a route towards the measurement of high-dimensional TMs either quickly or with access to limited numbers of measurements.

Nanobore fiber focus trap with enhanced tuning capabilities.

Confinement in fiber traps with two optical fibers facing one another relies on balancing the optical forces originating from the interaction of a scattering micro-object with the light beams delivered through the fibers. Here we demonstrate a novel type of dual fiber trap that involves the use of nanobore fibers, having a nano-channel located in the center of their fiber cores. This nano-element leads to a profound redistribution of the optical intensity and to considerably higher field gradients, yielding a trapping potential with greatly improved tuning properties compared to standard step-index fiber types. We evaluate the trap performance as a function of the fiber separation and find substantially higher stiffness for the nanobore fiber trap, especially in the range of short inter-fiber separations, while intermediate distances exhibit axial stiffness below that of the standard fiber. The results are in agreement with theoretical predictions and reveal that the exploitation of nanobore fibers allows for combinations of transverse and axial stiffness that cannot be accessed with common step-index fibers.

Label-free CARS microscopy through a multimode fiber endoscope.

Multimode fibres have recently been employed as high-resolution ultra-thin endoscopes, capable of imaging biological structures deep inside tissue in vivo. Here, we extend this technique to label-free non-linear microscopy with chemical contrast using coherent anti-Stokes Raman scattering (CARS) through a multimode fibre endoscope, which opens up new avenues for instant and in-situ diagnosis of potentially malignant tissue. We use a commercial 125 µm diameter, 0.29 NA GRIN fibre, and wavefront shaping on an SLM is used to create foci that are scanned behind the fibre facet across the sample. The chemical selectivity is demonstrated by imaging 2 µm polystyrene and 2.5 µm PMMA beads with per pixel integration time as low as 1 ms for epi-detection.

Wavelength dependent characterization of a multimode fibre endoscope.

Multimode fibres have recently shown promise as miniature endoscopic probes. When used for non-linear microscopy, the bandwidth of the imaging system limits the ability to focus light from broadband pulsed lasers as well as the possibility of wavelength tuning during the imaging. We demonstrate that the bandwidth is limited by the dispersion of the off-axis hologram displayed on the SLM, which can be corrected for, and by the limited bandwidth of the fibre itself. The selection of the fibre is therefore crucial for these experiments. In addition, we show that a standard prism pulse compressor is sufficient for material dispersion compensation for multi-photon imaging with a fibre endoscope.

Subcellular spatial resolution achieved for deep-brain imaging in vivo using a minimally invasive multimode fiber.

Achieving intravital optical imaging with diffraction-limited spatial resolution of deep-brain structures represents an important step toward the goal of understanding the mammalian central nervous system1-4. Advances in wavefront-shaping methods and computational power have recently allowed for a novel approach to high-resolution imaging, utilizing deterministic light propagation through optically complex media and, of particular importance for this work, multimode optical fibers (MMFs)5-7. We report a compact and highly optimized approach for minimally invasive in vivo brain imaging applications. The volume of tissue lesion was reduced by more than 100-fold, while preserving diffraction-limited imaging performance utilizing wavefront control of light propagation through a single 50-µm-core MMF. Here, we demonstrated high-resolution fluorescence imaging of subcellular neuronal structures, dendrites and synaptic specializations, in deep-brain regions of living mice, as well as monitored stimulus-driven functional Ca2+ responses. These results represent a major breakthrough in the compromise between high-resolution imaging and tissue damage, heralding new possibilities for deep-brain imaging in vivo.

High-fidelity multimode fibre-based endoscopy for deep brain in vivo imaging.

Progress in neuroscience relies on new techniques for investigating the complex dynamics of neuronal networks. An ongoing challenge is to achieve minimally invasive and high-resolution observations of neuronal activity in vivo inside deep brain areas. Recently introduced methods for holographic control of light propagation in complex media enable the use of a hair-thin multimode optical fibre as an ultranarrow imaging tool. Compared to endoscopes based on graded-index lenses or fibre bundles, this new approach offers a footprint reduction exceeding an order of magnitude, combined with a significant enhancement in resolution. We designed a compact and high-speed system for fluorescent imaging at the tip of a fibre, achieving a resolution of 1.18 ± 0.04 µm across a 50-µm field of view, yielding 7-kilopixel images at a rate of 3.5 frames/s. Furthermore, we demonstrate in vivo observations of cell bodies and processes of inhibitory neurons within deep layers of the visual cortex and hippocampus of anaesthetised mice. This study paves the way for modern microscopy to be applied deep inside tissues of living animal models while exerting a minimal impact on their structural and functional properties.

Robustness of Light-Transport Processes to Bending Deformations in Graded-Index Multimode Waveguides.

Light transport through a multimode optical waveguide undergoes changes when subjected to bending deformations. We show that optical waveguides with a perfectly parabolic refractive index profile are almost immune to bending, conserving the structure of propagation-invariant modes. Moreover, we show that changes to the transmission matrix of parabolic-index fibers due to bending can be expressed with only two free parameters, regardless of how complex a particular deformation is. We provide detailed analysis of experimentally measured transmission matrices of a commercially available graded-index fiber as well as a gradient-index rod lens featuring a very faithful parabolic refractive index profile. Although parabolic-index fibers with a sufficiently precise refractive index profile are not within our reach, we show that imaging performance with standard commercially available graded-index fibers is significantly less influenced by bending deformations than step-index types under the same conditions. Our work thus predicts that the availability of ultraprecise parabolic-index fibers will make endoscopic applications with flexible probes feasible and free from extremely elaborate computational challenges.