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.

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.

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.

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.

Comparison of nematic liquid-crystal and DMD based spatial light modulation in complex photonics.

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.

High-speed spatial control of the intensity, phase and polarisation of vector beams using a digital micro-mirror device.

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 ∼2 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.