A two-photon compound fiberscope to study the brain at all spatial and temporal scales.

Project Information

2P-BRAINSCOPY

Grant agreement ID: 101116227

DOI

10.3030/101116227

EC signature date 10 November 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 708 614,01

EU contribution

€ 1 708 614,00

Coordinated by

FUNDACIO INSTITUT DE BIOENGINYERIA DE CATALUNYA
Spain

Periodic Reporting for period 1 - 2P-BRAINSCOPY (A two-photon compound fiberscope to study the brain at all spatial and temporal scales.)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

Understanding how the brain gives rise to perception and behaviour is one of the most important challenges in modern science. The brain is composed of vast networks of interconnected neurons whose activity unfolds across space and time. To truly understand how these networks operate, scientists must be able to observe and influence the activity of many individual neurons across different spatial scales, while animals interact naturally with their environment.

Over the past decade, advanced optical methods have transformed neuroscience research. In particular, two-photon (2P) microscopy, combined with genetic strategies to label and control neurons, has enabled the visualization and manipulation of brain activity at single-cell resolution. Despite these advances, major technological limitations remain. Most existing approaches require animals to be immobilised, which restricts the study of natural behaviour. Furthermore, current microscopes cannot simultaneously provide the spatial resolution, imaging speed and large field of view needed to track how neural signals propagate across multiple brain regions. These constraints represent a significant barrier to addressing fundamental questions, such as how distant brain areas communicate, how sensory information is integrated, and how coordinated activity across large neuronal networks gives rise to behaviour. There is therefore a pressing need for new experimental tools.

The overall objective of this project is to overcome these technological barriers by developing a new generation of optical instruments for neuroscience. The project aims to create innovative 2P imaging and stimulation systems that combine high spatial resolution, fast temporal dynamics and large-scale coverage, while remaining compatible with freely moving animals. By providing access to brain activity across both local and brain-wide scales under natural conditions, the project delivers essential tools for understanding how patterns of neuronal activity generate perception and behaviour. In the longer term, these advances are expected to have a significant impact on fundamental brain research and to support future progress in the study of neurological and psychiatric disorders, where disrupted communication between brain regions plays a central role.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project builds upon our previous development of 2P-FENDO, an innovative miniaturised two-photon microscope designed for studying brain activity in freely moving mice. 2P-FENDO was the first optical system capable of performing 2P imaging and 2P photostimulation simultaneously through an optical fibre.

During the first two years of project, significant efforts were dedicated to consolidating these advances within the broader field of neurophotonics. This led to the publication of a review article presenting a critical perspective on the state of the art and future directions of multiphoton microscopy for neuronal imaging and photostimulation. The review highlighted current technological bottlenecks and outlined strategies for overcoming them, contributing to the scientific community’s understanding of emerging opportunities in the field.

Building on 2P-FENDO, we have recently achieved a major technological breakthrough with the development of a new proof-of-concept optical platform based on multiple fibre bundles. This system, named 2P-HOLMES, enables simultaneous access across spatially distant cortical regions, allowing to study distributed neuronal circuits. To protect this innovation, a patent application is currently being prepared, and the corresponding scientific results have been submitted for publication in a high-impact peer-reviewed journal of optics.

In parallel, in October 2024 we established a new research group on Nonlinear Photonics for Neuroscience at IBEC, Barcelona. We have dedicated large efforts on the recruitment of two doctoral students, a postdoctoral researcher and an engineer, providing the necessary expertise to develop and maintain advanced optical instrumentation and apply it to neuroscience. Through public tenders, we have acquired key equipment for the project, including an ultrafast laser system and relevant components for the construction of state-of-the-art 2P experimental platforms. Two major experimental efforts are currently underway: enhancing the performance of 2P-FENDO by increasing its field of view and imaging speed, and demonstrating a fully functional 2P-HOLMES setup capable of providing simultaneous optical access to neuronal activity across the majority of the mouse cortex.

Finally, we have established collaborations with (inter)national neuroscience research groups, enabling the integration and validation of the developed technologies into behavioural neuroscience studies and facilitating their adoption by the neuroscience community.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Existing 2P imaging and photostimulation technologies are constrained by limited spatial coverage and the need for head fixation, preventing simultaneous optical access to distributed neuronal circuits under natural behavioural conditions, and therefore remain insufficient to study large-scale communication across the brain.

A major advance achieved during the first two years of this project is the development of a novel multi-fibre optical system that enables parallel access to spatially distant brain regions. The resulting system, 2P-HOLMES, extends 2P microscopy with both imaging and photostimulation capabilities from the investigation of local circuits to the study of distributed cortical networks. By enabling simultaneous measurement and manipulation of neuronal activity across large cortical distances while preserving single-cell resolution, this approach overcomes a central limitation of existing technologies.

Beyond the specific instrument developed, 2P-HOLMES demonstrates how advanced beam shaping strategies combined with fibre-based light delivery can be used to scale 2P optical access without compromising spatial precision or temporal performance. These technological principles are broadly applicable and may inform future developments in multiphoton microscopy and other areas of optical instrumentation.

The potential impact of 2P-HOLMES lies in enabling neuroscientists to directly link distributed patterns of neuronal activity to behaviour, thereby supporting progress in systems neuroscience and brain-wide circuit analysis. In the longer term, such capabilities may contribute to improved understanding of neurological and psychiatric disorders, where dysfunctions in communication between brain regions are a defining feature.

At present, 2P-HOLMES is a proof-of-principle system. While it already enables simultaneous optical access to multiple cortical subregions, further work is required to extend this approach to provide coverage of the entire cortex and to validate performance in real behavioural contexts. Over the next three years, key objectives include increasing the number of accessible brain areas, improving system robustness and performance, and developing a compact and user-friendly implementation that can be readily disseminated in neuroscience laboratories. Achieving these goals will require continued technical development, experimental validation, and support for intellectual property management.