Periodic Reporting for period 1 - IVBM-4PAP (Development of an In-Vivo Brillouin Microscope (with application to Protein Aggregation-based Pathologies))
Reporting period: 2023-03-01 to 2024-02-29
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, either extrinsically induced or intracellularly generated, 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. Assessing the mechanical properties of living cells and tissues with similar spatiotemporal resolution in a non-invasive manner represents a major challenge. 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. Atomic force microscopy (AFM), the current gold standard in the field of mechanobiology, involves the application of nano-indenters to surfaces, e.g. the cellular membrane, to measure the quasi-static young’s modulus from the deflection of a cantilever. While this can provide high transverse spatial resolution on the nanometre scales, measurement is averaged along the contact (axial) direction (no 3D capability) and rely heavily on mechanical models to extract the mechanical parameters. Other approaches to measure elasticity include micropillar deformation, micropipette aspiration, deformability cytometry, magnetic twisting cytometry, optical tweezers and microdroplet deformation. All these techniques lack of spatial resolution capability and require direct contact to the cells of interest or rely on the introduction of foreign particles or do not work in multi–cellular situations. Other optical approaches, such as optical coherence elastography (OCE), requires external contact forces or ultrasound fields (US) to measure tissue displacements. While OCE enables rapid 3D imaging, it does not allow sub-cellular resolution, a limitation shared with other non-invasive techniques (US and NMRI). The device we will develop will be 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.
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 prodcue 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 current spectrometer, based on an interferometric filter for elastic peak suppression and a VIPA dispersive device, has been characterized and calibrated with reference samples. The setup is currently used to acuire the first Brilluoin map on samples of biological interest, and the first papers are in prepapration/submitted; ii) We have designed and realized a resonant cavity, with a mesured Q-factor just below 1000 with the non-linear optical crystal on board. The etherodyne optics and electronics have been purchased and, as foreseen, will be tested starting from the second project year.
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 signalling, tumour 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 analysing 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.
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 analysing 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.