Development of an In-Vivo Brillouin Microscope (with application to Protein Aggregation-based Pathologies)

Project Information

IVBM-4PAP

Grant agreement ID: 101098989

DOI

10.3030/101098989

EC signature date 16 December 2022

Start date 1 March 2023

End date 31 August 2027

Funded under

• The European Innovation Council (EIC)

Total cost

€ 3 868 938,00

EU contribution

€ 3 868 938,00

Coordinated by

FONDAZIONE ISTITUTO ITALIANO DI TECNOLOGIA
Italy

Periodic Reporting for period 2 - IVBM-4PAP (Development of an In-Vivo Brillouin Microscope (with application to Protein Aggregation-based Pathologies))

Reporting period: 2024-03-01 to 2025-02-28

Summary of the context and overall objectives of the project

The aim of the present project is to develop a fast-scan Brillouin Microscope capable of sub-millisecond acquisition time, therefore suitable for In-Vivo measurements in cells and tissues. In recent years, substantial progress has been made towards understanding how mechanical cues are transduced into biochemical signals to regulate multiple biological functions, both in physiological and pathological conditions. Biological systems can sense and decode a wide range of mechanical stimuli giving rise to multiple cellular responses, which can span several timescales. While molecular components can be routinely visualized in situ with powerful tools such as fluorescence microscopy, current approaches to measure cell mechanical properties in vivo have important limitations. To fill in this gap, we intend to develop a radically new instrument named In-Vivo Brillouin Microscope (IVBM) that will permit to measure the dynamic changes of mechanical properties of the living matter. Given the sub-micron resolution of IVBM, we will apply this technology to determine the viscoelastic properties of biomolecular condensates in multiple pathological settings. Existing methods to measure mechanical properties require the use of contact forces and lack appropriate sub-cellular resolution in 3D, or rely on the introduction of foreign particles or do not work in multi–cellular situations.
We will develop an add-on for microscopes like a laser scanning confocal head. The innovation relies on the RF cavities introduced to modulate electro-optically visible light to obtain stimulated Brillouin scattering and heterodyne detection. The advantages over current microscopy implementations of spontaneous Brillouin can be easily found in literature (TRL1), even though it has never been proven experimentally since no tuneable modulator in the GHZ range was available. Our test demonstrated the possibility to adopt RF cavities for this purpose (our starting point is TRL2) and we plan to validate experimentally this technology to reach TRL4.

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

As promised in the project, in the first year of activity we have i) sat up a standard instrument for benchmarking & ii) Designed and developed a resonant cavity. Beside these technical activities we have started to produce the biological samples for the test of the instrument. Specifically: i) The current setup present in the Crest Optics-IIT Joint-Lab laboratories has been upgraded with complementary microscopy techniques (fluorescence and transmitted light) necessary to perform conventional sample imaging. The setup is currently used to acquire the first Brillouin map on samples of biological interest, and the first papers are in preparation/submitted; ii) We have designed and realized a resonant cavity, with a measured Q-factor just below 1000 with the non-linear optical crystal on board. The heterodyne optics and electronics have been purchased and, as foreseen, will be tested starting from the second project year.
In the second year of the project we managed to solve one of the major limitations of Brillouin Microscopy, represented by spectral drifts, whose presence require continuous user intervention on the optical alignment of the spectrometer. To this aim, we introduced into the data acquisition workflow the signal coming from an Electro-Optic Modulator (EOM) (Figure 1): this implementation served simultaneously as a frequency reference, spectrometer calibrator, and temporal stabilizer of the measurements. We conducted several tests on this novel EOM-enhanced Brillouin Microscope, demonstrating its improved spectral stability in time compared to standard Brillouin Microscopes.
With this enhanced microscope, we acquired Brillouin data on protein condensates formed in vitro or over-expressed in cells, in collaboration with University of Zaragoza. Notably, we identified statistically significant differences between physiological (liquid-like) and pathogenic (solid-like) condensates solely on the basis of their Brillouin shift.
To address the challenge of imaging fast-moving condensates, we further modified the acquisition workflow by incorporating fluorescence-based tracking. Preliminary results indicate that this new method enables dynamic tracking of multiple condensates and real-time monitoring of their mechanical properties.

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)

By developing the IVBM, we plan to implement a gold standard instruments for biomechanical measurements, opening the way to many different clinical-oriented applications (tissues engineering, inter-cells signaling, tumor dissemination, chronic diseases, age-related diseases, ...).
With the development of IVBM we will introduce in the market a cost-effective instrument suitable to investigate mechanobiology in living cells and tissues and to demonstrate its power in two paradigmatic N&ND. The success of this program will have tremendous impact on the field of mechanobiology and biophotonics, providing an innovative technology whose application will result into societal and medical benefits for the affected patients and their relatives.
By defining the features of pathologically relevant biomolecular condensates, unveiling their mechanical properties in living cells and relate them with physiological parameters, we will make a crucial step towards the improvement of the diagnosis/prognosis of PTMPs and their responsiveness to therapeutic treatments. Specifically, developing patient-derived stem cell cultures and analyzing potential biomarkers by for specific diseases in the academic sector by the use of IVBM and then using those biomarkers in the clinical sector will open a new diagnostic path. On the treatment side, drug screening and development strategies using disease-specific cellular models and the IVBM platform will have a tremendous impact on patients and more broadly on the society as it will allow to define the most appropriate treatments for each specific patient, depending on the degree of measured mechano-alterations by IVBM. By combining the proposed stem cell-based disease models with IVBM, we will move in the direction of personalized medicine for a large set of patients affected by these chronic diseases.
The introduction of the EOM, finally, will pave the way for long-term, user-independent and high-precision biomechanical measurements in a fully automated workflow. This implementation is part of a preprint (DOI: 10.48550/arXiv.2412.20516) that will result in a scientific paper of next publication. We also successfully submitted a patent application (Pontecorvo, E. et al. SISTEMA PERFEZIONATO DI RIVELAZIONE BRILLOUIN 102024000005674. Italy patent (2024)).

Bone tissue slice of a murine Kabuki model imaged by different microscopy techniques.

Implementation of the Electro-Optic Modulator (EOM) in a standard Brillouin Microscope