Development of Brillouin Spectroscopy for Mechanotransduction Research

Lead Research Organisation: Newcastle University
Department Name: Institute of Genetic Medicine

Abstract

Mechanobiology is a rapidly developing area of science with the potential to improve significantly our knowledge of how cells function at a tissue/organ level. A better understanding of how cells react to their local mechanical environment (extracellular matrix, fluid flow, etc.) will also lead to advanced biomedical applications such as improved biomaterials and cell therapy. For such new therapeutic strategies, it is necessary to provide cells with a confined environment (niche) that enhances and regulates their proliferation and differentiation. In this regard, biomaterial technology currently leads the way in trying to fulfil these requirements by artificially recreating native-like three-dimensional environments. However, understanding how, why, and in what environment these cells differentiate in a lineage-specific manner is essential for understanding regenerative biology and developing better tissue engineering and stem cell therapy approaches. We believe that this underlying biology (i.e., the cell's responses to local mechanical stimuli) has not yet been properly investigated, due to a lack of appropriate tools, and that this is likely to undermine current attempts to i) understand mechanobiology; and ii) recreate the stem cell niche using purposeful biomaterials. Therefore, we plan to understand the role substrate stiffness plays in maintaining/directing cell phenotype at an unprecedented level by developing a sophisticated microscope that can quantify mechanical properties of the cells and their immediate environment whilst simultaneously measuring the cells' response to this environment at a gene and protein level. Our novel approach uses only light, and thus requires no direct physical contact with the sample and can probe deep into tissue structures. Most importantly, it can be performed under physiological conditions on live cells, facilitating real-time measurements under dynamic conditions. Such experiments have not previously been possible.
However, based on our recent success in building a confocal imaging system to measure tissue stiffness in 3D, we think this is now possible. Using the principle of Brillouin spectroscopy, the system detects light that has been scattered inelastically from acoustic phonons in the sample in a confocal optical arrangement to facilitate a non-contact, direct readout of the mechanical properties of tissues. We now wish to explore if this same confocal system can be used simultaneously to excite fluorescent molecules within the probed tissues. As such, our aims are to modify our existing machine so that confocal images of fluorescently-labelled proteins (from cells, tissues or tissue-engineered constructs) can be resolved and overlaid with simultaneous measurements of the mechanical properties of said cells/tissues. Ultimately, this will allow, for the first time and with unprecedented detail, the direct, real-time account of a cell's molecular response to differing local mechanical environments.

Technical Summary

Brillouin spectroscopy (BS) is a non-destructive, contact-free technique, which measures viscoelastic mechanical properties of materials. By measuring the frequency shift of light scattered inelastically via acoustic phonons one can deduce viscoelastic properties of the sample. By scanning the sample one can generate 3D maps of these properties.
However, while BS yields information on the viscoelasticity, it provides no information on the underlying molecular constituents at microscopic levels. Thus we wish to combine the technique with confocal fluorescence microscopy.
Our existing BS system will be modified by adding paths for fluorescence excitation and detection simultaneously and through the same Brillouin detection objective. Secondly, using established cell-based mechanotransduction models, the instrument's capabilities will be validated. We have previously demonstrated that collagen gels, with known and tractable stiffness, can be used to mimic natural extracellular matrix (ECM) in order to investigate the effect of substrate stiffness on stromal and epithelial stem cells. Our established models allow for the investigation of ECM stiffness in both 3D and 2D systems. The cells will be fluorescence-labelled against a panel of mechanotransduction-related proteins (e.g., cytokeratins, Yap/Taz, MAL, integrins). Furthermore, intracellular localisation of fluorescence-tagged recombinant mechanotransducers will be performed using stably-transfected corneal epithelial cell lines facilitating a dynamic mechanotransduction experiment that will enable known, precise changes in the mechanical environment to be plotted in real-time against changes in the mechanotransducer's intracellular localisation - the first time such an experiment will have been performed. Lastly, we plan to test our hypothesis that stem cell niches within adult tissues are normally more compliant than tissue surrounding the niche.

Planned Impact

The ubiquitous use of and critical need for imaging to study all aspects of microbial, plant, animal, and human biology cannot be understated. Because imaging provides spatiotemporal maps of the physical structure and the chemistry of biological and biomedical systems, it contributes to several of the major strategic initiatives in the UK. Antibiotic microbial resistance, food security, health and social impacts of an ageing population, improved treatments for cancer, and an understanding of the function and diseases in the brain will all be impacted and advanced by new capabilities in imaging-based measurement, modelling, diagnosis, and therapeutic intervention.

By combining the physical, life, and biomedical sciences we can foresee the application of the B/F microscope to reveal novel biochemical and physical structures and dynamics using approaches that have, to date, not been available. The potential impact of similar advances has recently been summarised in EPSRC's 'Healthcare Technologies Grand Challenges'' and again highlighted within the joint BBSRC, EPSRC, and MRC initiative "Technology Touching Life" (which ironically is a misnomer for our proposed technology as it works in a non-contact fashion).

The development of B/F microscopy for measuring biochemical and structural composition and environment in living tissues and organisms will be an important part of the future development of new discoveries and diagnostics. Moving forward, the development of such an imaging device that can be used to reveal the biological and biomedical structure and dynamics will be incredibly important for understanding the molecular basis of disease and the effect of candidate drugs on these diseases.

Non-academic beneficiaries will include the NHS, patients, UK regenerative medicine, and life sciences imaging businesses. Surgeons will gain improved predictability of clinical outcome following stem cell therapy (assuming success is affected by the stiffness of tissue into which the stem cell are transplanted), which would predictably lead to reduced repeat surgeries (lowering cost to NHS). Patients would benefit from improved diagnosis of diseases in which mechanotransduction has a known role in its pathology (e.g., cancer). This would happen following clinical trials and approximately 5 years from start of grant. Established healthcare companies can use the technology to move more towards the biomedical research space or to gain traction within the regenerative medicine space. This could be achieved within 3 years of the grant starting.
 
Description that it is feasible to combine brilloiun spectroscopy with confocal microscopy such that the biochemical and mechanical behviour of cells can be imaged side by side
Exploitation Route results will impact upon radily devloping area of Brilloiun spectroscopy within EU
Sectors Agriculture, Food and Drink,Electronics,Healthcare,Manufacturing, including Industrial Biotechology

 
Description clinical trial using stiffness as a tool for therapy is due to start summer 2020. mechanism of action elucidated by us using this microscope
First Year Of Impact 2018
Sector Healthcare
 
Title High Resolution Brillouin Spectroscopy for imaging tissue biomechanics 
Description Our work so far has focussed on building a High resolution Brillouin microscope that can probe collagen dense tissues or tissue-engineered constructs and measure their stiffness non-destructively (without physical perturbation). To this end, we have successfully built a new instrument and demonstrated its effectiveness in measuring discrete differences in the mechanical properties within biological samples (ranging from 20 µm-thin to 2 mm-thick) at a 1×1×5 µm resolution. The current instrument is capable of quickly generating highly reproducible data, using a novel adaptive optics inferferometric approach to suppress the elastic backscatter to measure Brillouin shifts with 0.01 GHz resolution, and with corresponding mechanical elastic sensitivity results comparable to those obtained in previous studies using different methodologies 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact Our High resolution Brillouin microscope has successfully facilitated a landmark study in which we show that the stem cell niche (within the cornea) is measurably more compliant that tissue outside the niche area and that this change in stiffness is directly associated with the stem cells level of differentiation. Moreover we show that by affecting tissue stiffness in vivo the cells within this area change their phenotype in a predicable manner. This works leads to a new therapeutic avenue. 
 
Description University of Missouri 
Organisation University of Missouri
Country United States 
Sector Academic/University 
PI Contribution we supplied the experimental design and intellectual prospective
Collaborator Contribution Prof Rajiv Mohan performed a series of animal studies in his lab invistigating the effect of collagenase as a drug for stem cell niche repair
Impact Publication in Nat Comms 2019 reporting on role of substrate stiffness on limbal stem cell niche function
Start Year 2016
 
Description Interviews for national news 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Press release associate with paper describing new way to grow human corneas then led to 2x TV news interviews (BBC and ITV) and BBC Radio NE
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/news/uk-england-tyne-41685982