High-resolution line-scan Brillouin microscopy for live imaging of mechanical properties during embryo development.

Brillouin microscopy can assess mechanical properties of biological samples in a three-dimensional (3D), all-optical and hence non-contact fashion, but its weak signals often lead to long imaging times and require an illumination dosage harmful for living organisms. Here, we present a high-resolution line-scanning Brillouin microscope for multiplexed and hence fast 3D imaging of dynamic biological processes with low phototoxicity. The improved background suppression and resolution, in combination with fluorescence light-sheet imaging, enables the visualization of the mechanical properties of cells and tissues over space and time in living organism models such as fruit flies, ascidians and mouse embryos.

Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens.

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump-probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Direct Three-Dimensional Measurement of Refractive Index via Dual Photon-Phonon Scattering.

We developed a microscopy technique that can measure the local refractive index without sampling the optical phase delay of the electromagnetic radiation. To do this, we designed and experimentally demonstrated a setup with two colocalized Brillouin scattering interactions that couple to a common acoustic phonon axis; in this scenario, the ratio of Brillouin frequency shifts depends on the refractive index, but not on any other mechanical and/or optical properties of the sample. Integrating the spectral measurement within a confocal microscope, the refractive index is mapped at micron-scale three-dimensional resolution. As the refractive index is probed in epidetection and without assumptions on the geometrical dimensions of the sample, this method may prove useful to characterize biological cells and tissues.

Intensity noise coupling in soliton fiber oscillators.

We present an experimental and numerical study on the spectrally resolved pump-to-output intensity noise coupling in soliton fiber oscillators. In our study, we observe a strong pump noise coupling to the Kelly sidebands, while the coupling to the soliton pulse is damped. This behavior is observed in erbium-doped as well as holmium-doped fiber oscillators and confirmed by numerical modeling. It can be seen as a general feature of laser oscillators in which soliton pulse formation is dominant. We show that spectral blocking of the Kelly sidebands outside the laser cavity can improve the intensity noise performance of the laser dramatically.

Age-progressive interplay of HSP-proteostasis, ECM-cell junctions and biomechanics ensures C. elegans astroglial architecture.

Tissue integrity is sensitive to temperature, tension, age, and is sustained throughout life by adaptive cell-autonomous or extrinsic mechanisms. Safeguarding the remarkably-complex architectures of neurons and glia ensures age-dependent integrity of functional circuits. Here, we report mechanisms sustaining the integrity of C. elegans CEPsh astrocyte-like glia. We combine large-scale genetics with manipulation of genes, cells, and their environment, quantitative imaging of cellular/ subcellular features, tissue material properties and extracellular matrix (ECM). We identify mutants with age-progressive, environment-dependent defects in glial architecture, consequent disruption of neuronal architecture, and abnormal aging. Functional loss of epithelial Hsp70/Hsc70-cochaperone BAG2 causes ECM disruption, altered tissue biomechanics, and hypersensitivity of glia to environmental temperature and mechanics. Glial-cell junctions ensure epithelia-ECM-CEPsh glia association. Modifying glial junctions or ECM mechanics safeguards glial integrity against disrupted BAG2-proteostasis. Overall, we present a finely-regulated interplay of proteostasis-ECM and cell junctions with conserved components that ensures age-progressive robustness of glial architecture.

Mechanical mapping of mammalian follicle development using Brillouin microscopy.

In early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find 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. Our work provides an alternative approach to study the role of mechanics in follicle morphogenesis and might pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

A 3D Brillouin microscopy dataset of the in-vivo zebrafish eye.

In this work we present three-dimensional (3D) measurements of Brillouin scattering spectra of the in-vivo zebrafish larvae eye. This dataset was obtained by Brillouin microscopy, an emerging all-optical and non-contact technique that gives access to material properties through the process of Brillouin scattering. Herein, we share a representative 3D dataset of spectral properties of 48-52 h post-fertilization (hpf) zebrafish embryos. These spectral properties can be related to a complex longitudinal modulus and thus elastic and viscous properties given knowledge of refractive index and material density. The dataset encompasses the crystalline lens as well as several different retinal layers. This data provides a valuable resource as well as a starting point for researchers interested in the mechanobiology of vertebrate eye development.

Intestinal intermediate filament polypeptides in C. elegans: Common and isotype-specific contributions to intestinal ultrastructure and function.

The abundance and diversity of intermediate filaments (IFs) in the C. elegans intestine indicate important contributions to intestinal function and organismal wellbeing. Fluorescent IF reporters localize below the actin-rich brush border and are highly enriched in the lumen-enveloping endotube, which is attached to the C. elegans apical junction. Mapping intestinal viscoelasticity by contact-free Brillouin microscopy reveals that the IF-rich endotube is positioned at the interface between the stiff brush border and soft cytoplasm suggesting a mechanical buffering function to deal with the frequent luminal distortions occurring during food intake and movement. In accordance, depletion of IFB-2, IFC-2 and IFD-2 leads to intestinal lumen dilation although depletion of IFC-1, IFD-1 and IFP-1 do not. Ultrastructural analyses of loss of function mutants further show that IFC-2 mutants have a rarefied endotube and IFB-2 mutants lack an endotube altogether. Remarkably, almost all IFB-2- and IFC-2-deficient animals develop to fertile adults. But developmental retardation, reduced brood size, altered survival and increased sensitivity to microbial toxin, osmotic and oxidative stress are seen in both mutants albeit to different degrees. Taken together, we propose that individual intestinal IF polypeptides contribute in different ways to endotube morphogenesis and cooperate to cope with changing environments.

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

In this work, we quantify the mechanical properties of the extra-cellular matrix (ECM) in live zebrafish using Brillouin microscopy. Optimization of the imaging conditions and parameters, combined with careful spectral analysis, allows us to resolve the thin ECM and distinguish its Brillouin frequency shift, a proxy for mechanical properties, from the surrounding tissue. High-resolution mechanical mapping further enables the direct measurement of the thickness of the ECM label-free and in-vivo. We find the ECM to be ~500 nm thick, and in very good agreement with electron microscopy quantification. Our results open the door for future studies that aim to investigate the role of ECM mechanics for zebrafish morphogenesis and axis elongation.