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.

• Keywords
• fiber connection, freely moving, holographic endoscopy, in-vivo imaging, multicore fiber, multimode fiber,
• Publication type
• Journal Article MeSH

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.

• Keywords
• complex media photonics, deep brain imaging, endoscope, in-vivo imaging, multimode fiber, wavefront shaping,
• Publication type
• Journal Article MeSH

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.

• MeSH
• Head * MeSH
• Microscopy, Confocal MeSH
• Brain * MeSH
• Mice MeSH
• Neurons MeSH
• Blood Flow Velocity MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• Publication type
• Journal Article MeSH

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.

• Keywords
• blood flow, fluorescence, label free, molecular sensors, multimodal, optical imaging, optogenetics,
• Publication type
• Journal Article MeSH

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.

• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• Publication type
• Journal Article MeSH