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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.
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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.
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Diagnóstico por Imagem , CalibragemRESUMO
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.
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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.
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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.
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Endoscopia/métodos , Tecnologia de Fibra Óptica/instrumentação , Holografia/métodos , Microscopia/métodos , Imagens de Fantasmas , Fibras ÓpticasRESUMO
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.
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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.
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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.
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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.
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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.
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Light-sheet microscopy facilitates rapid, high-contrast, volumetric imaging with minimal sample exposure. However, the rapid divergence of a traditional Gaussian light sheet restricts the field of view (FOV) that provides innate subcellular resolution. We show that the Airy beam innately yields high contrast and resolution up to a tenfold larger FOV. In contrast to the Bessel beam, which also provides an increased FOV, the Airy beam's characteristic asymmetric excitation pattern results in all fluorescence contributing positively to the contrast, enabling a step change for light-sheet microscopy.
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Microscopia/instrumentação , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Simulação por Computador , Desenho de Equipamento , Corantes Fluorescentes/química , Luz , Microscopia/métodos , Microscopia de Fluorescência/métodos , Microesferas , Distribuição Normal , Óptica e Fotônica , Espalhamento de Radiação , Peixe-ZebraRESUMO
Digital micro-mirror devices (DMDs) have recently emerged as practical spatial light modulators (SLMs) for applications in photonics, primarily due to their modulation rates, which exceed by several orders of magnitude those of the already well-established nematic liquid crystal (LC)-based SLMs. This, however, comes at the expense of limited modulation depth and diffraction efficiency. Here we compare the beam-shaping fidelity of both technologies when applied to light control in complex environments, including an aberrated optical system, a highly scattering layer and a multimode optical fibre. We show that, despite their binary amplitude-only modulation, DMDs are capable of higher beam-shaping fidelity compared to LC-SLMs in all considered regimes.
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The dynamic spatial control of light fields is essential to a range of applications, from microscopy to optical micro-manipulation and communications. Here we describe the use of a single digital micro-mirror device (DMD) to generate and rapidly switch vector beams with spatially controllable intensity, phase and polarisation. We demonstrate local spatial control over linear, elliptical and circular polarisation, allowing the generation of radially and azimuthally polarised beams and Poincaré beams. All of these can be switched at rates of up to 4kHz (limited only by our DMD model), a rate â¼2 orders of magnitude faster than the switching speeds of typical phase-only spatial light modulators. The polarisation state of the generated beams is characterised with spatially resolved Stokes measurements. We also describe detail of technical considerations when using a DMD, and quantify the mode capacity and efficiency of the beam generation. The high-speed switching capabilities of this method will be particularly useful for the control of light propagation through complex media such as multimode fibers, where rapid spatial modulation of intensity, phase and polarisation is required.
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Real-time, on-demand, beam shaping at the end of the multimode fiber has recently been made possible by exploiting the computational power of rapidly evolving graphics processing unit (GPU) technology [Opt. Express 22, 2933 (2014)]. However, the current state-of-the-art system requires the presence of an acousto-optic deflector (AOD) to produce images at the end of the fiber without interference effects between neighboring output points. Here, we present a system free from the AOD complexity where we achieve the removal of the undesired interference effects computationally using GPU implemented Gerchberg-Saxton and Yang-Gu algorithms. The GPU implementation is two orders of magnitude faster than the CPU implementation which allows video-rate image control at the distal end of the fiber virtually free of interference effects.
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Gráficos por Computador , Computadores , Holografia/instrumentação , Fibras Ópticas , Acústica , AlgoritmosRESUMO
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.
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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.
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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.
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Encéfalo , Cabeça , Camundongos , Animais , Microscopia Confocal , Velocidade do Fluxo Sanguíneo , NeurôniosRESUMO
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.
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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.
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We present a powerful approach towards full understanding of laser light propagation through multimode optical fibres and control of the light at the fibre output. Transmission of light within a multimode fibre introduces randomization of laser beam amplitude, phase and polarization. We discuss the importance of each of these factors and introduce an experimental geometry allowing full analysis of the light transmission through the multimode fibre and subsequent beam-shaping using a single spatial light modulator. We show that using this approach one can generate an arbitrary output optical field within the accessible field of view and range of spatial frequencies given by fibre core diameter and numerical aperture, respectively, that contains over 80% of the total available power. We also show that this technology has applications in biophotonics. As an example, we demonstrate the manipulation of colloidal microparticles.