STED-enabled super-resolution multimode-fibre based holographic endoscopy for deep-tissue observations of neuronal connectivity

Informations projet

STEDGate

N° de convention de subvention: 101158010

DOI

10.3030/101158010

Projet clôturé

Date de signature de la CE 25 Mars 2024

Date de début 1 Mai 2024

Date de fin 31 Octobre 2025

Financé au titre de

• European Research Council (ERC)

Coût total

Aucune donnée

Contribution de l’UE

€ 150 000,00

Coordonné par

LEIBNIZ-INSTITUT FUER PHOTONISCHE TECHNOLOGIEN E.V.
Germany

Periodic Reporting for period 1 - STEDGate (STED-enabled super-resolution multimode-fibre based holographic endoscopy for deep-tissue observations of neuronal connectivity)

Période du rapport: 2024-05-01 au 2025-10-31

Résumé du contexte et des objectifs généraux du projet

Neurological disorders have emerged as a significant global societal burden, exemplified by afflictions like Alzheimer's and Parkinson's, impacting over one billion individuals globally and surpassing the combined economic burden of cancer and diabetes. This has spurred a concerted global effort, with increased support for neuroscience research. These disorders often target deep brain regions and profoundly influence the structural connectivity of neuronal cells within functional circuits. Synapses, where neurons exchange information, exhibit plasticity, altering information transmission efficiency, shape, and position. Understanding the mechanisms underlying these structural changes, especially in neuronal circuits, remains limited in both healthy and affected individuals. The ERC PoC project STEDGate seeks to advance our understanding of neuronal connectivity and plasticity by developing STED-enabled holographic endo-nanoscopy for neuroscience. This ground-breaking technology promises atraumatic nanoscale in vivo imaging of deep brain structures reaching depths up to 5 mm beneath the brain's surface. Collaborating with the start-up endeavour DeepEn, the team aims to facilitate the commercial transition of this technology. Making deep-tisue nanoscopy available globally will revolutionize our ability to monitor and understand neurological disorders and, ultimately, offer new avenues for intervention and treatment.
The objectives for the projects are:
- Construct of the empowered STED-enabled holographic nanoscope, operating at the highest possible performance allowed by contemporary commercially available technology.
- Achieve repeated observations of the same structures.
- Apply the technology to monitor the structural connectivity of neuronal circuits within the living brain.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

We developed a fully in-vivo–compatible STED endo-nanoscopy system that integrates advances in fibre-probe engineering, control software, and in-vivo imaging methodology.
- A side-view multimode fibre probe was engineered and iteratively refined based on in-vivo feedback, resulting in improved surface quality, reproducibility, photon collection efficiency, spatial resolution, and reduced tissue disruption.
- Image-acquisition and control routines were upgraded, including synchronisation with oximetry-based heart-rhythm signals to suppress heartbeat-induced motion artefacts.
- A new platform for chronic imaging was established, enabling re-deployment of the same animal and repeated measurements over multiple sessions.
A key technical challenge was the broadening of excitation laser pulses at higher power levels. During the project, a laser source with uniquely suitable specifications (pulse duration, spectral purity) became available on the market. We negotiated a one-month, free-of-charge loan of this system, which allowed us to validate the improved resolving power directly in in-vivo experiments.
The integrated system was validated in anaesthetized Thy1-GFP mice, demonstrating robust resolution enhancement, controlled photobleaching, and minimal photo-damage in both cortical and hippocampal regions (see Fig. 1). The chronic-imaging configuration supported reliable multi-day observations, opening opportunities for studying the evolution of neuronal connectivity and synaptic plasticity. In addition, we implemented a dual-channel in-vivo imaging mode by repurposing the depletion laser to excite a second fluorophore, enabling visualization of complementary structures.
To permit longitudinal tracking of synapses we opted for a viral system to deliver a fusion protein of a synaptic scaffolding protein (PSD95) and GFP (PSD95-GFP).
The transfected animal model has been evaluated for its suitability based on imaging of post-mortem tissue sections, both with confocal microscopy and the STED-enabled endo-nanoscopy. The tests have revealed that further optimization of the sparseness of expression of PSD95-GFP is needed.
The results have been reflected in two patent applications (one published, one under examination). These will be followed by a publication disseminating the results across the Neuroscience community.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The most prominent impact of the technology is in monitoring the synaptic plasticity across deep-brain structures. This is critical because these areas govern learning, memory, motivation, and emotional regulation, and are the primary sites affected in neurological and psychiatric disorders such as Alzheimer’s disease, Parkinson’s disease, depression, and addiction. Accessing these synapses at nanoscale resolution will allow researchers to understand how microscopic changes drive behaviour and disease progression. Most importantly, it will enable the development and efficacy verification of new therapies, disease combat strategies and interventions.
To transfer our technology into commercially viable product portfolio, there are several additional aspects in need of addressing. While the technology has been proven applicable with this project, the full range of applicability remains to be fully explored. There are numerous thinkable genetic labels and their combinations enabling addressing of diverse neuroscience questions. A few case-studies of successful deployments in diverse areas of neuroscience research can greatly enhance the market readiness.
In relation to the previous aspect, the application workflow wouldn't end when images become available. The analysis of the image data is a slow and tedious process for human, and urgently calls for the deployment of modern data processing approaches involving machine-learning and AI.

Beyond the initially proposed research agenda, we have identified that the developed optical path meets all requirements for other imaging modalities, most importantly the time-domain modality of the Fluorescence-lifetime imaging microscopy (FLIM). FLIM provides quantitative information on the molecular environment and metabolic state of brain cells as well as precise monitoring of cellular functions and activity beyond conventional intensity-based imaging. We have therefore included first performance assessment of this modality into the project's agenda (see Fig. 2).

Figure 2: In-vivo demonstration of FLIM modality (dendrites and blood vessels).

Figure 1: In-vivo demonstration of resolution enhancement (dendrites in cortex).