Brillouin microscopy: an emerging tool for mechanobiology.

The role and importance of mechanical properties of cells and tissues in cellular function, development and disease has widely been acknowledged, however standard techniques currently used to assess them exhibit intrinsic limitations. Recently, Brillouin microscopy, a type of optical elastography, has emerged as a non-destructive, label- and contact-free method that can probe the viscoelastic properties of biological samples with diffraction-limited resolution in 3D. This led to increased attention amongst the biological and medical research communities, but it also sparked debates about the interpretation and relevance of the measured physical quantities. Here, we review this emerging technology by describing the underlying biophysical principles and discussing the interpretation of Brillouin spectra arising from heterogeneous biological matter. We further elaborate on the technique's limitations, as well as its potential for gaining insights in biology, in order to guide interested researchers from various fields.

Dark-field Brillouin microscopy.

Brillouin microscopy is a non-contact and label-free technique for mapping fundamental micro-mechanical properties in the volume of biological systems. Specular reflections and elastic scattering easily overwhelm the weak Brillouin spectra due to the limited extinction of virtually imaged phased array spectrometers, thereby affecting the image acquisition. In this Letter, a dark-field method was demonstrated to reject the elastic background light using an annular illumination and a confocal detection. To validate the method, images of polystyrene and liquid samples were obtained using both a confocal and the dark-field system. An extinction ratio of 30 dB was readily achieved.

Birefringence-induced phase delay enables Brillouin mechanical imaging in turbid media.

Acoustic vibrations of matter convey fundamental viscoelastic information that can be optically retrieved by hyperfine spectral analysis of the inelastic Brillouin scattered light. Increasing evidence of the central role of the viscoelastic properties in biological processes has stimulated the rise of non-contact Brillouin microscopy, yet this method faces challenges in turbid samples due to overwhelming elastic background light. Here, we introduce a common-path Birefringence-Induced Phase Delay (BIPD) filter to disentangle the polarization states of the Brillouin and Rayleigh signals, enabling the rejection of the background light using a polarizer. We demonstrate a 65 dB extinction ratio in a single optical pass collecting Brillouin spectra in extremely scattering environments and across highly reflective interfaces. We further employ the BIPD filter to image bone tissues from a mouse model of osteopetrosis, highlighting altered biomechanical properties compared to the healthy control. Results herald new opportunities in mechanobiology where turbid biological samples remain poorly characterized.

Digistain: a digital staining instrument for histopathology.

We describe a new mid-infrared (mid-IR) imaging method specifically designed to augment the H + E tissue staining protocol. Images are taken with bespoke IR filters at wavelengths that enable chemical maps to be generated, corresponding to the cytoplasmic (amide) and nuclear (phosphodiester) components of unstained oesophageal tissue sections. A suitably calibrated combination of these generates false colour computer images that reproduce not only the tissue morphology, but also accurate and quantitative distributions of the nuclear-to-cytoplasmic ratio throughout the tissue section. This parameter is a well documented marker of malignancy, and because the images can be taken and interpreted by clinically trained personnel in a few seconds, we believe this new "digistain" approach makes spectroscopic mid-IR imaging techniques available for the first time as a practical, specific and sensitive augmentation to standard clinical cancer diagnosis methods.

Recent progress and current opinions in Brillouin microscopy for life science applications.

Many important biological functions and processes are reflected in cell and tissue mechanical properties such as elasticity and viscosity. However, current techniques used for measuring these properties have major limitations, such as that they can often not measure inside intact cells and/or require physical contact-which cells can react to and change. Brillouin light scattering offers the ability to measure mechanical properties in a non-contact and label-free manner inside of objects with high spatial resolution using light, and hence has emerged as an attractive method during the past decade. This new approach, coined "Brillouin microscopy," which integrates highly interdisciplinary concepts from physics, engineering, and mechanobiology, has led to a vibrant new community that has organized itself via a European funded (COST Action) network. Here we share our current assessment and opinion of the field, as emerged from a recent dedicated workshop. In particular, we discuss the prospects towards improved and more bio-compatible instrumentation, novel strategies to infer more accurate and quantitative mechanical measurements, as well as our current view on the biomechanical interpretation of the Brillouin spectra.

Scattering Assisted Imaging.

Standard imaging systems provide a spatial resolution that is ultimately dictated by the numerical aperture (NA) of the illumination and collection optics. In biological tissues, the resolution is strongly affected by scattering, which limits the penetration depth to a few tenths of microns. Here, we exploit the properties of speckle patterns embedded into a strongly scattering matrix to illuminate the sample at high spatial frequency content. Combining adaptive optics with a custom deconvolution algorithm, we obtain an increase in the transverse spatial resolution by a factor of 2.5 with respect to the natural diffraction limit. Our Scattering Assisted Imaging (SAI) provides an effective solution to increase the resolution when long working distance optics are needed, potentially paving the way to bulk imaging in turbid tissues.

Quantifying cellular forces and biomechanical properties by correlative micropillar traction force and Brillouin microscopy.

Cells sense and respond to external physical forces and substrate rigidity by regulating their cell shape, internal cytoskeletal tension, and stiffness. Here we show that the combination of micropillar traction force and noncontact Brillouin microscopy provides access to cell-generated forces and intracellular mechanical properties at optical resolution. Actin-rich cytoplasmic domains of 3T3 fibroblasts showed significantly higher Brillouin shifts, indicating a potential increase in stiffness when adhering on fibronectin-coated glass compared to soft PDMS micropillars. Our findings demonstrate the complementarity of micropillar traction force and Brillouin microscopy to better understand the relation between cell force generation and the intracellular mechanical properties.

3D models in the new era of immune oncology: focus on T cells, CAF and ECM.

Immune checkpoint inhibitor therapy has changed clinical practice for patients with different cancers, since these agents have demonstrated a significant improvement of overall survival and are effective in many patients. However, an intrinsic or acquired resistance frequently occur and biomarkers predictive of responsiveness should help in patient selection and in defining the adequate treatment options. A deep analysis of the complexity of the tumor microenvironment is likely to further advance the field and hopefully identify more effective combined immunotherapeutic strategies. Here we review the current knowledge on tumor microenvironment, focusing on T cells, cancer associated fibroblasts and extracellular matrix. The use of 3D cell culture models to resemble tumor microenvironment landscape and to screen immunomodulatory drugs is also reviewed.

Mechanical Durotactic Environment Enhances Specific Glioblastoma Cell Responses.

Background: A hallmark of glioblastoma is represented by their ability to widely disperse throughout the brain parenchyma. The importance of developing new anti-migratory targets is critical to reduce recurrence and improve therapeutic efficacy. Methods: Polydimethylsiloxane substrates, either mechanically uniform or presenting durotactic cues, were fabricated to assess GBM cell morphological and dynamical response with and without pharmacological inhibition of NNMII contractility, of its upstream regulator ROCK and actin polymerization. Results: Glioma cells mechanotactic efficiency varied depending on the rigidity compliance of substrates. Morphologically, glioma cells on highly rigid and soft bulk substrates displayed bigger and elongated aggregates whereas on durotactic substrates the same cells were homogeneously dispersed with a less elongated morphology. The durotactic cues also induced a motility change, cell phenotype dependent, and with cells being more invasive on stiffer substrates. Pharmacological inhibition of myosin or ROCK revealed a rigidity-insensitivity, unlike inhibition of microfilament contraction and polymerization of F-actin, suggesting that alternative signalling is used to respond to durotactic cues. Conclusions: The presence of a distinct mechanical cue is an important factor in cell migration. Together, our results provide support for a durotactic role of glioma cells that acts through actomyosin contractility to regulate the aggressive properties of GBM cells.

Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) has been genetically linked to mutations in RNA-binding proteins (RBPs), including FUS. Here, we report the RNA interactome of wild-type and mutant FUS in human motor neurons (MNs). This analysis identified a number of RNA targets. Whereas the wild-type protein preferentially binds introns, the ALS mutation causes a shift toward 3' UTRs. Neural ELAV-like RBPs are among mutant FUS targets. As a result, ELAVL4 protein levels are increased in mutant MNs. ELAVL4 and mutant FUS interact and co-localize in cytoplasmic speckles with altered biomechanical properties. Upon oxidative stress, ELAVL4 and mutant FUS are engaged in stress granules. In the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. We propose that pathological mutations in FUS trigger an aberrant crosstalk with ELAVL4 with implications for ALS.

Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS.

Altered cellular biomechanics have been implicated as key photogenic triggers in age-related diseases. An aberrant liquid-to-solid phase transition, observed in in vitro reconstituted droplets of FUS protein, has been recently proposed as a possible pathogenic mechanism for amyotrophic lateral sclerosis (ALS). Whether such transition occurs in cell environments is currently unknown as a consequence of the limited measuring capability of the existing techniques, which are invasive or lack of subcellular resolution. Here we developed a non-contact and label-free imaging method, named background-deflection Brillouin microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling, to the best of our knowledge, the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our findings provide fundamental insights on the critical aggregation step underlying the neurodegenerative ALS disease.

Cancellation of Bessel beam side lobes for high-contrast light sheet microscopy.

An ideal illumination for light sheet fluorescence microscopy entails both a localized and a propagation invariant optical field. Bessel beams and Airy beams satisfy these conditions, but their non-diffracting feature comes at the cost of the presence of high-energy side lobes that notably degrade the imaging contrast and induce photobleaching. Here, we demonstrate the use of a light droplet illumination whose side lobes are suppressed by interfering Bessel beams of specific k-vectors. Our droplet illumination readily achieves more than 50% extinction of the light distributed across the Bessel side lobes, providing a more efficient energy localization without loss in transverse resolution. In a standard light sheet fluorescence microscope, we demonstrate a two-fold contrast enhancement imaging micron-scale fluorescent beads. Results pave the way to new opportunities for rapid and deep in vivo observations of large-scale biological systems.

Biomechanics of subcellular structures by non-invasive Brillouin microscopy.

Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p < 0.0001) and the surrounding cytoplasm (p < 0.0001). Moreover, we demonstrate the mechanical response of cells to Latrunculin-A, a drug that reduces cell stiffness by preventing cytoskeletal assembly. Our technique can therefore generate valuable insights into cellular biomechanics and its role in pathophysiology.

Quantification of plaque stiffness by Brillouin microscopy in experimental thin cap fibroatheroma.

Plaques vulnerable to rupture are characterized by a thin and stiff fibrous cap overlaying a soft lipid-rich necrotic core. The ability to measure local plaque stiffness directly to quantify plaque stress and predict rupture potential would be very attractive, but no current technology does so. This study seeks to validate the use of Brillouin microscopy to measure the Brillouin frequency shift, which is related to stiffness, within vulnerable plaques. The left carotid artery of an ApoE(-/-)mouse was instrumented with a cuff that induced vulnerable plaque development in nine weeks. Adjacent histological sections from the instrumented and control arteries were stained for either lipids or collagen content, or imaged with confocal Brillouin microscopy. Mean Brillouin frequency shift was 15.79 ± 0.09 GHz in the plaque compared with 16.24 ± 0.15 (p < 0.002) and 17.16 ± 0.56 GHz (p < 0.002) in the media of the diseased and control vessel sections, respectively. In addition, frequency shift exhibited a strong inverse correlation with lipid area of -0.67 ± 0.06 (p < 0.01) and strong direct correlation with collagen area of 0.71 ± 0.15 (p < 0.05). This is the first study, to the best of our knowledge, to apply Brillouin spectroscopy to quantify atherosclerotic plaque stiffness, which motivates combining this technology with intravascular imaging to improve detection of vulnerable plaques in patients.