Since the start of the ERC grant, we have made substantial progress towards its main goals as outlined above. We have designed and built a Brillouin microscope based on selective line-illumination geometry (termed LSBM) and demonstrated multiplexed signal acquisition at high spatial resolution, thus significantly speeding up image acquisition by ~100-fold (C. Bevilacqua et al., Nature Methods, in press). This methodological advancement now makes comprehensive, large-volume 3D mechanical measurements possible in living tissues and organisms. Furthermore, our LSBM imaging system features a fluorescence light-sheet modality which allows correlating the measured mechanical properties to the underlying molecular constituents by means of active labelling of e.g. cell nuclei or membranes.
In an effort to increase the intrinsically weak Brillouin scattering signal, we have also started work on so-called Stimulated Brillouin Spectroscopy (SBS), which promises to drastically increase signal levels by many orders of magnitude. Since typical SBS approaches require laser illumination levels that are prohibitively high for many applications in biology, we developed a pulsed-SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction. With this, we could decrease the required pump laser powers ~20-fold without affecting the signal levels or spectral precision. This then enabled live-imaging of photo-sensitive single cells, zebrafish larvae, mouse embryos and adult C. elegans (F. Yang et al, bioRxiv: 2022.11.10.515835 (2022), in review at Nature Methods).
On the data analysis side, we have developed powerful methods based on GPU-acceleration (
https://github.com/prevedel-lab/brillouin-gpu-acceleration(se abrirá en una nueva ventana)). Our GPU-enhanced numerical fitting routine is tailored to the lineshape of our experimental spectra and allows to analyse the multiplexed (100+) spectra of our LSBM in real-time and with high precision. Overall, our analysis pipeline yields a >1000-fold enhancement in processing time over standard methods employed in the field. Our LSBM and SBS systems and analysis pipeline thus form a powerful and unprecedented technological basis for our ongoing and future work to study the role of mechanical properties in tissue morphogenesis and animal development.
While developing these advanced BM methodologies, we have utilized a self-built confocal Brillouin microscopy to shed first light on the role of mechanics during mammalian development. Here, we have quantified the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using our Brillouin tools. We found that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation (C. Chan et al., Comms Bio. (2021)). Furthermore, in a collaboration with colleagues at EMBL, our BM tools were instrumental to reveal how tissue visco-elasticity is affecting glia integrity and architecture in C. Elegans in an age-dependent fashion (Coraggio et al, submitted).
All combined, our work provides novel methods and approaches to study the role of mechanics in animal development. We are thus well on track with respect to the aims and goals envisioned in our original proposal.
