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.

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.

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.