LENIMBRA final report

Using an optical fiber as an objective lens and reaching the ultimate miniaturization of endo-microscopic imaging tools

Functional neuron imaging in living animals requires minimally-invasive imaging tools capable of two-photon excited fluorescence imaging: A persistent challenge in two-photon excited fluorescence (2PEF) imaging is deep imaging in tissue. In particular, a challenge lies in recording neuronal activity with single-cell resolution deep in the living brain. 2PEF microscopy is a method of choice for imaging the activity of neurons but due to tissue scattering, its imaging depth is limited to 100 μm - 1 mm.
Responding to this challenge, the LENIMBRA project proposes to develop methodology allowing to acquire a 2PEF image at the tip of a wave guide - a multi-core fiber. This wave guide will constitute the flexible probe of a « lensless endoscope ».
The objective is to develop a 2PEF lensless endoscope having a flexible probe with diameter 200 μm, small enough to permit deep insertion into the living mouse brain for deep tissue image acquisition.
The project has several novel aspects. The multi-core fibers developed will be the first ones optimized for 2PEF imaging. The endoscope developed represents the uncharted territory of nonlinear imaging through a waveguide. Its use in conjunction with existing neuroimaging methods represents a novel way of probing long-range functional connections in the brain.
The results obtained during LENIMBRA will enable other nonlinear image contrasts and methods in endoscopes, opening even more new horizons in brain imaging.

Specialty optical fiber, fiber micro-structuring, and wave front shaping methods combine to give an ultra-miniaturized imaging tool : the « lensless endoscope »: To fulfill the objective, an interdisciplinary approach will be taken. The instrumental and methodological development will be guided by a targeted neuroimaging experiment, simultaneous imaging of neuronal activity in the hippocampus and entorhinal cortex of a living mouse. Multi-core fibers will be designed and fabricated explicitly for the project. To realize the lensless endoscope, imaging through multi-core fibers will be achieved through an integrality of interferometry, holography, wave front shaping, and adaptive optics.
The 2PEF lensless endoscope will acquire images a few 100 μm from the tip of its probe, Fig. 1. Then, the imaging properties will be the same, no matter how deep the probe is inserted into tissue. The probe is a wave guide - a multi-core fiber whose diameter is reduced to the fundamental limit, the diameter of the wave guide itself, minimizing damage done upon insertion. The small size enables using the endoscope simultaneously with other micro- or macro-scale neuro-imaging methods.

Key results encourage future work on lensless endoscopes : We have designed new specialty optical fibers and have demonstrated how these designs can overcome the principal technical challenge : assuring that the acquired images are not distorted by fiber movement. We also have encouraging results on how micro-structuring of the fiber tip can increase the spatial resolution of the lensless endoscope. And finally, we have succeeded in acquiring images of neurons in brain slices from transgenic mice whose parameters closely resemble the targeted in vivo application.
Three patent applications filed in collaboration with the technology transfer accelerator SATT-SudEst have led to a technology maturation project. The established ties with the SATT SudEst will be upheld.
The project has strengthened the interdisciplinary collaborations between the participating laboratories, and follow-on projects are envisioned.

Scientific production: The LENIMBRA project has resulted in a scientific production of 10 scientific articles, including one review article, as well as 15 contributions at international conferences, including one invited talk.
Several inventions have also been made during the course of the project, which have led to three patent applications. These patents now constitute a cluster of patents protecting the intellectual property behind the developed lensless endoscope technology.

Figure 1. Concepts of the project.

 

Factual information: The project «Lensless endoscopy : imaging deep in the living brain » (LENIMBRA) is a fundamental research project coordinated by Esben Ravn Andresen at the Institut Fresnel, Marseille. The following laboratories have taken part in the project : Institut de Recherche sur les Composants logiciels et matériels pour l'Information et la Communication Avancée (IRCICA), Lille and Institut de Neurobiologie de la Méditerranée (INMED), Marseille). The project started in September 2014 and lasted 48 months. The ANR financed the project with 251 680 €.

See also the LENIMBRA publication list.