# 2P-FENDO-II: A fiber bundle microscope for all-optical, large field-of-view brain studies in freely moving mice

## TL;DR

A new microscope allows high-resolution imaging and control of brain activity in freely moving mice over large areas.

## Contribution

2P-FENDO-II improves field of view and optical performance for all-optical studies in freely moving mice.

## Key findings

- 2P-FENDO-II achieves a four-times-larger field of view and better homogeneity for imaging and photostimulation.
- The system enables concomitant calcium imaging and optogenetic control in multiple brain regions of freely moving mice.
- Complex shape illumination targeting dendrites is demonstrated in naturalistic exploration settings.

## Abstract

All-optical strategies enable identification of functional neuronal ensembles with calcium imaging and replay/alter their spatiotemporal activity with optogenetics to decipher their behavioral implications. We previously developed a fiber-coupled microscope enabling two-photon (2P) functional imaging and 2P holographic photostimulation with near-single-cell resolution in freely moving mice: 2P-FENDO. Here, we present a significantly optimized 2P-FENDO-II system that achieves a four-times-larger field of view and a more homogeneous light distribution across the field of view, both for imaging and photostimulation, while achieving better flexibility and thus optimal adaptation to the study of freely moving mice. We demonstrate the performance and versatility of 2P-FENDO-II in experiments targeting the somatosensory cortex, the visual cortex, or the cerebellar cortex, in which we show concomitant calcium imaging with jGCaMP7s and optogenetic control with ChRmine. These enhancements establish 2P-FENDO-II as a groundbreaking tool for all-optical interrogation of neuronal circuits on large volume in naturalistic situations.

•2P-FENDO-II enables two-photon all-optical study in freely moving mice over a large FOV•A miniaturized objective reduces optical aberrations•Leached fibers enhance image homogeneity and flexibility for natural exploration•We achieved complex shape illumination to target dendrites in freely moving mice

2P-FENDO-II enables two-photon all-optical study in freely moving mice over a large FOV

A miniaturized objective reduces optical aberrations

Leached fibers enhance image homogeneity and flexibility for natural exploration

We achieved complex shape illumination to target dendrites in freely moving mice

Understanding how neuronal activity gives rise to behavior is a central question in neuroscience. Recent advances in miniaturized multiphoton scanning heads have enabled the development of two-photon miniscopes capable of imaging brain activity at cellular resolution during freely moving behavior. While impressive, this technology still lacks the ability to perform all-optical experiments in which neuronal circuits can be simultaneously imaged and manipulated with cellular precision, as is possible under head-fixed conditions. To address this limitation, we previously developed a fiber-bundle-based endoscope, the 2P-FENDO, coupled to GRIN lenses, and demonstrated fast two-photon imaging and optogenetic manipulation in freely moving mice. Here, we present an improved version of the system that achieves a larger and more homogeneous field of view, as well as increased flexibility.

Studying the brain’s natural mechanisms requires tools to precisely manipulate widespread neuronal circuits in freely moving animals. Blot et al. present a new microscope that allows the recording and control of neural activity with high precision, down to the level of individual cells, across large brain areas during naturalistic behavior.