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.
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.
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.
Using multiphoton microscopy, we can image the tissue with sub-cellular resolution at depths down to 1.5 mm. Beyond this reach, the light must be delivered to the target region by optical relay elements inserted into the tissuethe endoscopes. Focusing light through a step-index multimode optical fibre (MMF) using wave-front control enables minimally-invasive endoscopy. With fibre micro-endoscopes, we can reach any depth with negligible tissue disruption and keep the resolution below 1 micrometre, sufficient for in vivo microscopy. We designed a novel custom-terminated multimode fibre probe. This work will focus on optical fibre probe manufacturing methods (etching, polishing and coating) and their impact on imaging quality.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.