Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy

Context

It is becoming increasingly clear that mechanical cues are important for cell survival, behavior and fate determination [1]. Our understanding of these mechanical cues, however, is far from complete due to the difficulty to measure and perturb them in a precise manner in vivo. While techniques such as atomic force microscopy [2], micropipette aspiration [3], or optical tweezers [4] have been instrumental in measuring mechanical forces, they are invasive techniques and consequently their application for in vivo measurements is limited. By using the frequency shift observed in scattered light, Brillouin microscopy offers an alternative non-invasive optical microscopy technique to measure material properties in living cells and tissues [5,6].

What is Brillouin microscopy (a short technical background)

In 1922, Léon Brillouin discovered that light is scattered by inherent density fluctuations in materials that are induced by thermally-driven collective oscillations of atoms and/or molecules (acoustic phonons).  This scattering is inelastic and thereby induces a tiny frequency shift between the incident and scattered light. Brillouin microscopy measures this frequency shift. The Brillouin frequency shift has been shown to be related to the longitudinal elastic modulus (inverse compressibility) of the imaged material and its density (Eq. 1). Furthermore, the shift depends on the refractive index and parameters controlled by the microscope setup such as the angle of scattering and the light wavelength. As Brillouin microscopy relies directly on the material property of biological structures and tissues imaged, it is label-free.

 

 

 

Key findings and why I chose this preprint

For the first time, Brillouin microscopy has been shown to measure the thickness of ECM in vivo in the zebrafish notochord with high resolution (Fig. 1). The authors nicely com­pare this measurement to thickness measurements obtained with electron microscopy. Both imaging modalities agree.  As Brillouin microscopy is label-free, it does not interfere with fluorescent imaging and therefore provides a complementary imaging modality to confocal or light sheet imaging. Consequently, Brillouin microscopy opens up the door for new, complex experi­ments.

In this paper, the authors also point out that Brillouin microscopy is subject to the challenge of many optical systems, i.e. the anisotropy of the system point spread function (PSF). Keeping this in mind is important for the interpretation of the resulting material property map.

 

 

Future directions and interesting follow-up questions

• How do the measurements of ECM thickness vary over development and along the anterior-posterior axis?
• Pioneering work of determining ECM components have been done by Palombo et al. [8]. Can the authors determine from their measurements different ECM components, potentially also over development?
• There is an ongoing debate whether the longitudinal elastic modulus is important for biological processes, for example whether it is correlated to the Young’s modulus that is a measure of the stiffness of a material [6,7]. Is the Young’s modulus and thus stiffness in ECM related to the longitudinal elastic modulus?
• Currently, the exposure time is 0.25 seconds per pixel. Can the acquisition time be accelerated?

 

References

[1]  Bloom, A. B., & Zaman, M. H. (2014). Influence of the microenvironment on cell fate determination and migration. Physiological genomics.
[2] Krieg, M., Fläschner, G., Alsteens, D., Gaub, B. M., Roos, W. H., Wuite, G. J. L., et al. (2018). Atomic force microscopy-based mechanobiology. Nature Reviews Physics.
[3] Guevorkian, K., & Maitre, J.-L. (2017). Micropipette aspiration: A unique tool for exploring cell and tissue mechanics in vivo. Methods Cell Biol.
[4] Killian, J. L., Ye, F., & Wang, M. D. (2018). Optical Tweezers: A Force to Be Reckoned With. Cell.
[5] Scarcelli, G., & Yun, S. H. (2007). Confocal Brillouin microscopy for three-dimensional mechanical imaging. Nature photonics.
[6] Schlussler, R., Mollmert, S., Abuhattum, S., Cojoc, G., Muller, P., Kim, K., et al. (2018). Mechanical Mapping of Spinal Cord Growth and Repair in Living Zebrafish Larvae by Brillouin Imaging. Biophysical Journal.
[7] Wu, P.-J., Kabakova, I. V., Ruberti, J. W., Sherwood, J. M., Dunlop, I. E., Paterson, C., et al. (2018). Water content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materials. Nature Methods.
[8] Palombo, F., Winlove, C. P., Edginton, R. S., Green, E. , Stone,  N., Caponi, S., Madami, M.  & Fioretto, D. (2014). Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering. Journal of The Royal Society, Inteface.

 

 

 

 

Tags: brillouin microscopy, ecm, label-free, microscopy, zebrafish

doi: https://doi.org/10.1242/prelights.7023

Read preprint (1 votes)