Hair-thin confocal fluorescence endo-microscopy for deep-brain in-vivo imaging

TL;DR

This paper introduces a novel MMF-based endoscope with confocal filtering capabilities, significantly improving deep-brain in-vivo fluorescence imaging contrast and resolution by rejecting out-of-focus light.

Contribution

It presents a new composite fibre probe combining graded-index and step-index MMFs to enable confocal-like imaging in deep-brain endoscopy.

Findings

Enhanced image contrast and resolution in deep-brain imaging

Successful visualization of neuronal structures and calcium signals in vivo

Demonstrated potential for minimally invasive, high-resolution brain imaging

Abstract

Confocal and multi-photon microscopy are widely used for in-vivo fluorescence imaging of biological tissues such as the brain, offering non-invasive access up to ~1 mm depth without major loss in performance. A recently-developed alternative is holographic endoscopy, which exploits controlled light transport through hair-thin optical fibres. With minimal invasiveness, it provides observations at comparable spatial resolution, while extending its applicability to unprecedented depths. It has been used to resolve details of sub-cellular structural connectivity, record neuronal signalling, and monitor blood flow from the deepest locations of the living brain. Yet, its use, particularly in densely labelled brain regions, has so far been constrained by significant contrast loss, primarily due to the absence of a practical mechanism for rejecting out-of-focus fluorescence light -- a capability inherently provided by confocal and multi-photon microscopy. Exploring opportunities in the structure of light modes of different MMF types we identify the possibility of achieving an analogue to confocal fluorescence microscopy through MMF-based endoscopes. Using a novel composite fibre probe that combines graded-index and step-index MMFs, we enable spatially resolved signal collection and selective rejection of out-of-focus light. This confocal filtering significantly enhances image contrast and resolution by suppressing background and off-plane signals. We demonstrate improved imaging performance on fine structural connectivity and intracellular calcium signalling in living mouse brain.