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

Carlo Bevilacqua, Héctor Sánchez Iranzo, Dmitry Richter, Alba Diz-Muñoz, Robert Prevedel

Posted on: 8 January 2019 , updated on: 9 January 2019

Preprint posted on 10 December 2018

Article now published in Biomedical Optics Express at http://dx.doi.org/10.1364/boe.10.001420

How to measure ECM in living organisms with high-resolution but without labels? A new preprint from the lab of Diz-Muñoz and @Prevedel_Lab gives an answer:

Selected by Stephan Daetwyler

Categories: bioengineering, biophysics

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.

Equation 1: Relationship of Brillouin frequency shift to material properties

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.

**Fig. 1: Confocal imaging and Brillouin map of 3dpf zebrafish notochord (from Fig. 3 A,B of the preprint)** While **(A)** fluorescence confocal imaging (sheath cells, GFP) is selective , Brillouin microscopy **(B)** measures the material property of all structures. It allowed the identification of e.g. ECM surrounding the sheath cells (s) as long stripes (dark red), the vacuole (v) of the vacuolated cells, muscle cells (m) or surrounding water (w).

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

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Author's response

Robert Prevedel and Alba Diz-Muñoz shared

How do the measurements of ECM thickness vary over development and along the anterior-posterior axis?

This is a great question! Anisotropies in ECM stiffness have been shown to be relevant for follicle elongation in Drosophila melanogaster [1], another epithelial system under expanding pressure encased by a similar ECM layer. Whether and how ECM thickness and/or mechanics vary over development and thus contribute to axis elongation is the focus of ongoing work between our labs, but at the moment it’s too early to make serious conclusions.

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?

The structure and composition of the zebrafish notochord’s ECM has been previously assessed: it is formed by three layers, a thin inner laminin-rich basement membrane layer [2], a middle layer of densely packed collagen fibers (especially rich in collagen type-II which is also a major constituent of cartilage [3]), and an outer layer in which extracellular fibers run perpendicularly to the middle layer [4]. Distinguishing their composition from their spectra would certainly be challenging with Brillouin scattering and require significant improvements in spectral resolution.

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?

There is no formal relation, as these elastic moduli (Young’s and the longitudinal) have different definitions. The main difference is that the longitudinal modulus probes the ratio of uniaxial stress to strain in a confined condition, i.e. in which the material is not allowed to expand sideways, thereby changing its density and/or volume. The ‘stiffness’ measured by Brillouin scattering is therefore fundamentally different from Young’s modulus, which requires the volume to be kept constant. As a consequence, although both moduli share the same units (Pa), the longitudinal modulus probed in Brillouin scattering is in general much higher (~GPa). One can see them as complementary mechanical parameters which both are related to ‘stiffness’.

As we often get this sort of questions from biologist, we have recently written a (hopefully) accessible Review on this topic. It’s now on ArXiv (http://arxiv.org/abs/1901.02006) and in fact it would be great to get feedback on this from the community!

Currently, the exposure time is 0.25 seconds per pixel. Can the acquisition time be accelerated?

In principle yes, but in practice not without trade-offs, which typically go hand-in-hand with poorer signal-to-noise and hence spectral precision. There are a number of approaches different to ours to speed this up, most notably by nonlinear stimulated [5] and impulsive [6] Brillouin modalities or line-scanning [7] approaches. While these are in principle promising, more work is needed to turn them into truly live-imaging modalities for biological specimens. Again, our Review has a Section devoted to the technical state-of-art for interested readers.

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