Periodic Reporting for period 2 - Brillouin4Life (Development of advanced optical tools for studying cellular mechanics at high spatial and temporal resolution)
Période du rapport: 2022-03-01 au 2023-08-31
suffer from very low speed, high phototoxicity, and difficulties in quantification, thus prohibiting meaningful investigations in the life sciences.
In the context of this interdisciplinary ERC grant, my group will develop unique and innovative optical imaging technologies based on BM to overcome its current drawbacks and establish it as a revolutionary tool for live tissue and cellular biophysics studies. In particular, we will work towards a highly-multiplexed BM with selective-plane illumination to maximize speed, resolution and depth penetration, while minimizing photodamage (Aim 1). At the same time, we will combine BM with other imaging modalities to obtain correlative datasets and to accurately quantify and interpret the measured mechanical properties (Aim 2). We will then apply these methodological advancements together with fellow biologists to study the role of elasticity in tissue morphogenesis and animal development, thereby contributing to a better understanding of the role of biomechanics in developmental biology (Aim 3).
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). 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.