Lensless endoscopy: Imaging deep in the living brain – LENIMBRA

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.

Expected results:

* A series of multi-core fibers optimiwed for 2PEF imaging.

* Stabilisation algorithms that eliminate practical challenges (elimination of fiber bend-induced phase drifts, elimination of fiber bend-induced group delay drift).

* Brain-fiber interface.

* A functioning lensless endoscope with a flexible probe, capable of 2PEF imaging.

* Demonstration of in-vivo endoscopic imaging in two distant brain regions.

In view of the preliminary results, it can be expected that the results from LENIMBRA will enable several known nonlinear image contrast in endoscopes. First time around three-photon excited fluorescence (3PEF), second-harmonic generation (SHG), and third-harmonic generation (THG) imaging which are not conceptually very different from 2PEF imaging. Next, also multi-beam contrast mechanisms such as coherent anti-Stokes Raman scattering (CARS) and stimulated-Raman scattering (SRS), and two-color 2PEF.

LENIMBRA's approach of a hybrid microscope-endoscope in in vivo imaging of the hippocampus-entorhinal cortex system can surely be specialized to also study other important, yet deep, regions of the brain and their interactions with more shallow regions in the cortex or the hippocampus.

Electroencephalography (EEG), magnetoencephalography (MEG), and functional magnetic resonance imaging (fMRI) are the principal techniques for measuring brain activity at the scale of the whole brain. They only indirectly report neuron activity and cannot achieve single-cell resolution. The lensless endoscope developed during LENIMBRA should be sufficiently non-invasive that it can be used in conjunction also with all these methods, so as to build a bridge between the macro-level and the fundamental, neuronal circuit level.

At the single-cell level, local field recording tools exist which directly measure extracellular electrical signals with excellent temporal resolution, however, without any spatial resolution. The lensless endoscope could be used in conjunction with local field recording, giving a complementarity in spatial and temporal resolution.

Optogenetics is hailed as a critical future technology for neuroscience. In the same way that optogenetic methods can be performed under a 2PEF microscope, LENIMBRA's 2PEF endoscope will also have the capability with all that it entails.

[1] E. R. Andresen, S. Sivankutty, G. Bouwmans, L. Gallais, S. Monneret, H. Rigneault, «Measurement and compensation of residual group delay in a multi-core fiber for lensless endoscopy«, J. Opt. Soc. Am. B 32(6), 1221-1228 (2015).

[2] E. R. Andresen, S. Sivankutty, G. Bouwmans, O. Vainvinq, L. Gallais, S. Monneret, H. Rigneault, «Towards two-photon lensless endoscopes: inter-core group delay compensation in a multi-core fiber «, Proc. SPIE 9536, Advanced Microscopy Techniques IV; and Neurophotonics II, 953605 (2015).

[3] S. Sivankutty, E. R. Andresen, R. Cossart, G. Bouwmans, S. Monneret, H. Rigneault, «Ultra-thin rigid endoscope: Two-photon imaging through a graded-index multi-mode fiber«, arXiv preprint arXiv:1510.04818 (2015)

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 microns - 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 - with the same speed and flexibility as a 2PEF microscope. 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 microns, small enough to permit deep insertion into the living mouse brain for deep tissue image acquisition.
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 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.