SignificanceHair-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.AimWe 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.ApproachWe 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.ResultsWe 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.ConclusionsThis 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.
Neuroscience research related to functionality, connectivity and metabolism of neuronal circuits, individual neuronal cells and sub-cellular structures, nowadays, experiences a burgeoning need to develop techniques for the detailed investigation inside the complexity of the living matter. Particularly, high-resolution observations combined with an extended depth of penetration in tissue represents an ongoing challenge.
Holographic control of light propagation in complex media opens a promising way to overcome this technological barrier via exploiting multimode fibres as hair-thin, minimally-invasive endoscopes. This concept allows for more than one order of magnitude reduction of the instrument’s footprint and a significant enhancement of imaging resolution, compared with current minimally invasive endoscopes.
Here, we demonstrate a compact and high-speed system for fluorescent imaging at the tip of a fibre. The instrument’s performance reaches micron-scale resolution across a field of view 50 micrometres, yielding 7-kilopixel image information at a rate of 3.5 frames per second. The resolution limit is dictated only by the numerical aperture of the fibre probe, and the contrast/pureness of the focal points, utilised for raster-scanning regime, approach the theoretical limits for phase-only holographic wavefront shaping.
The achieved performance allowed for in-vivo observations of neuronal somata and processes, residing deep inside the visual cortex and hippocampus of an animal model with minimal damage to the tissue surrounding the fibre penetration area.
We believe that this demonstration represents an important step towards implementations of various advanced forms of imaging through multimode fibre based endoscopes to address numerous key challenges in neuroscience.
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