Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Subclinical Keratoconus Detection and Characterization Using Motion-Tracking Brillouin Microscopy.

PURPOSE: To characterize focal biomechanical alterations in subclinical keratoconus (SKC) using motion-tracking (MT) Brillouin microscopy and evaluate the ability of MT Brillouin metrics to differentiate eyes with SKC from normal control eyes. DESIGN: Prospective cross-sectional study. PARTICIPANTS: Thirty eyes from 30 patients were evaluated, including 15 eyes from 15 bilaterally normal patients and 15 eyes with SKC from 15 patients. METHODS: All patients underwent Scheimpflug tomography and MT Brillouin microscopy using a custom-built device. Mean and minimum MT Brillouin values within the anterior plateau region and anterior 150 µm were generated. Scheimpflug metrics evaluated included inferior-superior (IS) value, maximum keratometry (Kmax), thinnest corneal thickness, asymmetry indices, Belin/Ambrosio display total deviation, and Ambrosio relational thickness. Receiver operating characteristic (ROC) curves were generated for all Scheimpflug and MT Brillouin metrics evaluated to determine the area under the ROC curve (AUC), sensitivity, and specificity for each variable. MAIN OUTCOME MEASURES: Discriminative performance based on AUC, sensitivity, and specificity. RESULTS: No significant differences were found between groups for age, sex, manifest refraction spherical equivalent, corrected distance visual acuity, Kmax, or KISA% index. Among Scheimpflug metrics, significant differences were found between groups for thinnest corneal thickness (556 µm vs. 522 µm; P < 0.001), IS value (0.29 diopter [D] vs. 1.05 D; P < 0.001), index of vertical asymmetry (IVA; 0.10 vs. 0.19; P < 0.001), and keratoconus index (1.01 vs. 1.05; P < 0.001), and no significant differences were found for any other Scheimpflug metric. Among MT Brillouin metrics, clear differences were found between control eyes and eyes with SKC for mean plateau (5.71 GHz vs. 5.68 GHz; P < 0.0001), minimum plateau (5.69 GHz vs. 5.65 GHz; P < 0.0001), mean anterior 150 µm (5.72 GHz vs. 5.68 GHz; P < 0.0001), and minimum anterior 150 µm (5.70 GHz vs. 5.66 GHz; P < 0.001). All MT Brillouin plateau and anterior 150 µm mean and minimum metrics fully differentiated groups (AUC, 1.0 for each), whereas the best performing Scheimpflug metrics were keratoconus index (AUC, 0.91), IS value (AUC, 0.89), and IVA (AUC, 0.88). CONCLUSIONS: Motion-tracking Brillouin microscopy metrics effectively characterize focal corneal biomechanical alterations in eyes with SKC and clearly differentiated these eyes from control eyes, including eyes that were not differentiated accurately using Scheimpflug metrics. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

A Brillouin microscopy analysis of the crystalline lenses of Chinese adults with myopia.

To evaluate lenticular biomechanical and geometric parameters in Chinese adults with myopia and identify relevant factors using Brillouin microscopy (BM) and Pentacam. The biomechanical and geometric properties of the ocular lenses of Chinese adults with myopia were quantified using BM. Anterior segment images were acquired using a Pentacam. Correlated factors including age, sex, spherical equivalent (SE), intraocular pressure (IOP), axial length (AL), white-to-white ratio (WTW), central corneal thickness (CCT), anterior chamber depth (ACD), anterior chamber volume (ACV), and anterior chamber angle (ACA) were analyzed. We studied 65 eyes from 65 participants (mean age, 25.23 ± 6.12 years). Width of Top Plateau (WTP), Width of Bottom Plateau (WBP), Slope of Anterior Cortex (SAC), Slope of Posterior Cortex (SPC), and Height of Plateau (Height) metrics obtained using BM showed mean values of 2.597 ± 0.393 mm, 4.310 ± 0.535 mm, 1.344 ± 0.549 GPa/mm, -1.343 ± 0.480 GPa/mm, and 3.373 ± 0.048 GPa, respectively. No significant correlation was found between these parameters and sex, SE, IOP, CCT, ACA, or Height. Interestingly, WBP (r = 0.467, P < 0.001), SAC (r = 0.412, P = 0.001), and SPC (r = -0.280, P = 0.024) were significantly associated with age, and an age-related increase of WBP (slope of 35.36 ± 10.08 µm per year) was identified. Both ACD and ACV showed significant correlations with SAC (r = 0.329 and 0.380, P = 0.008 and 0.002, respectively), but not with SPC. BM provided a novel perspective on lenticular biomechanical and geometric properties in Chinese adults with myopia, which correlated with age, AL, WTW, ACD, and ACV.

Bile Is a Selective Elevator for Mucosal Mechanics and Transport.

Mucus mechanically protects the intestinal epithelium and impacts the absorption of drugs, with a largely unknown role for bile. We explored the impacts of bile on mucosal biomechanics and drug transport within mucus. Bile diffused with square-root-of-time kinetics and interplayed with mucus, leading to transient stiffening captured in Brillouin images and a concentration-dependent change from subdiffusive to Brownian-like diffusion kinetics within the mucus demonstrated by differential dynamic microscopy. Bile-interacting drugs, Fluphenazine and Perphenazine, diffused faster through mucus in the presence of bile, while Metoprolol, a drug with no bile interaction, displayed consistent diffusion. Our findings were corroborated by rat studies, where co-dosing of a bile acid sequestrant substantially reduced the bioavailability of Perphenazine but not Metoprolol. We clustered over 50 drugs based on their interactions with bile and mucin. Drugs that interacted with bile also interacted with mucin but not vice versa. This study detailed the dynamics of mucus biomechanics under bile exposure and linked the ability of a drug to interact with bile to its abbility to interact with mucus.

Corneal biomechanics: Measurement and structural correlations.

The characterization of corneal biomechanical properties has important implications for the management of ocular disease and prediction of surgical responses. Corneal refractive surgery outcomes, progression or stabilization of ectatic disease, and intraocular pressure determination are just examples of the many key clinical problems that depend highly upon corneal biomechanical characteristics. However, to date there is no gold standard measurement technique. Since the advent of a 1-dimensional (1D) air-puff based technique for measuring the corneal surface response in 2005, advances in clinical imaging technology have yielded increasingly sophisticated approaches to characterizing the biomechanical properties of the cornea. Novel analyses of 1D responses are expanding the clinical utility of commercially-available air-puff-based instruments, and other imaging modalities-including optical coherence elastography (OCE), Brillouin microscopy and phase-decorrelation ocular coherence tomography (PhD-OCT)-offer new opportunities for probing local biomechanical behavior in 3-dimensional space and drawing new inferences about the relationships between corneal structure, mechanical behavior, and corneal refractive function. These advances are likely to drive greater clinical adoption of in vivo biomechanical analysis and to support more personalized medical and surgical decision-making.

Nuclear Mechanics within Intact Cells Is Regulated by Cytoskeletal Network and Internal Nanostructures.

The mechanical properties of the cellular nucleus are extensively studied as they play a critical role in important processes, such as cell migration, gene transcription, and stem cell differentiation. While the mechanical properties of the isolated nucleus have been tested, there is a lack of measurements about the mechanical behavior of the nucleus within intact cells and specifically about the interplay of internal nuclear components with the intracellular microenvironment, because current testing methods are based on contact and only allow studying the nucleus after isolation from a cell or disruption of cytoskeleton. Here, all-optical Brillouin microscopy and 3D chemomechanical modeling are used to investigate the regulation of nuclear mechanics in physiological conditions. It is observed that the nuclear modulus can be modulated by epigenetic regulation targeting internal nuclear nanostructures such as lamin A/C and chromatin. It is also found that nuclear modulus is strongly regulated by cytoskeletal behavior through a robust mechanism conserved in different culturing conditions. Given the active role of cytoskeletal modulation in nearly all cell functions, this work will enable to reveal highly relevant mechanisms of nuclear mechanical regulations in physiological and pathological conditions.

Dual optical elastography detects TGF - ß -induced alterations in the biomechanical properties of skin scaffolds.

Significance: The skin's mechanical properties are tightly regulated. Various pathologies can affect skin stiffness, and understanding these changes is a focus in tissue engineering. Ex vivo skin scaffolds are a robust platform for evaluating the effects of various genetic and molecular interactions on the skin. Transforming growth factor-beta ( TGF - ß ) is a critical signaling molecule in the skin that can regulate the amount of collagen and elastin in the skin and, consequently, its mechanical properties. Aim: This study investigates the biomechanical properties of bio-engineered skin scaffolds, focusing on the influence of TGF - ß , a signaling molecule with diverse cellular functions. Approach: The TGF - ß receptor I inhibitor, galunisertib, was employed to assess the mechanical changes resulting from dysregulation of TGF - ß . Skin scaffold samples, grouped into three categories (control, TGF - ß -treated, and TGF - ß + galunisertib-treated), were prepared in two distinct culture media-one with aprotinin (AP) and another without. Two optical elastography techniques, namely wave-based optical coherence elastography (OCE) and Brillouin microscopy, were utilized to quantify the biomechanical properties of the tissues. Results: Results showed significantly higher wave speed (with AP, p < 0.001 ; without AP, p < 0.001 ) and Brillouin frequency shift (with AP, p < 0.001 ; without AP, p = 0.01 ) in TGF - ß -treated group compared with the control group. The difference in wave speed between the control and TGF - ß + galunisertib with ( p = 0.10 ) and without AP ( p = 0.36 ) was not significant. Moreover, the TGF - ß + galunisertib-treated group exhibited lower wave speed without and with AP and reduced Brillouin frequency shift than the TGF - ß -treated group without AP, further strengthening the potential role of TGF - ß in regulating the mechanical properties of the samples. Conclusions: These findings offer valuable insights into TGF - ß -induced biomechanical alterations in bio-engineered skin scaffolds, highlighting the potential of OCE and Brillouin microscopy in the development of targeted therapies in conditions involving abnormal tissue remodeling and fibrosis.

Mechanical evolution of metastatic cancer cells in three-dimensional microenvironment.

Cellular biomechanics plays critical roles in cancer metastasis and tumor progression. Existing studies on cancer cell biomechanics are mostly conducted in flat 2D conditions, where cells' behavior can differ considerably from those in 3D physiological environments. Despite great advances in developing 3D in vitro models, probing cellular elasticity in 3D conditions remains a major challenge for existing technologies. In this work, we utilize optical Brillouin microscopy to longitudinally acquire mechanical images of growing cancerous spheroids over the period of eight days. The dense mechanical mapping from Brillouin microscopy enables us to extract spatially resolved and temporally evolving mechanical features that were previously inaccessible. Using an established machine learning algorithm, we demonstrate that incorporating these extracted mechanical features significantly improves the classification accuracy of cancer cells, from 74% to 95%. Building on this finding, we have developed a deep learning pipeline capable of accurately differentiating cancerous spheroids from normal ones solely using Brillouin images, suggesting the mechanical features of cancer cells could potentially serve as a new biomarker in cancer classification and detection.

Properties of dentin, enamel and their junction, studied with Brillouin scattering and compared to Raman microscopy.

OBJECTIVE: Dentin, enamel and the transition zone, called the dentin-enamel junction (DEJ), have an organization and properties that play a critical role in tooth resilience and in stopping the propagation of cracks. Understanding their chemical and micro-biomechanical properties is then of foremost importance. The aim of this study is to apply Brillouin microscopy on a complex biological structure, that is, the DEJ, and to compare these results with those obtained with Raman microscopy. DESIGN: Both techniques allow noncontact measurements at the microscopic scale. Brillouin microscopy is based on the interaction between acoustic phonons and laser photons and gives a relation between the frequency shift of the scattered light and the stiffness of the sample. Raman spectra contain peaks related to specific chemical bonds. RESULTS: Comparison of the Brillouin and Raman cartographies reveals correlations between mechanical and chemical properties. Indeed, the shapes of the phosphate content and stiffness curves are similar. The two spectroscopies give compatible values for the mean distance between two tubules, i.e., 4-6 µm. Moreover, for the first time, the daily cross striations of enamel could be studied, indicating a relationship between the variation in the phosphate concentration and the variation in the rigidity within the enamel prisms. CONCLUSIONS: We demonstrate here the possibility of using Brillouin scattering microscopy to both study complex biological materials such as the enamel-dentin junction and visualize secondary structures. Correlations between the chemical composition and mechanical properties could help in better understanding the tissue histology.

Non-contact and label-free biomechanical imaging: Stimulated Brillouin microscopy and beyond.

Brillouin microscopy based on spontaneous Brillouin scattering has emerged as a unique elastography technique because of its merit of non-contact, label-free, and high-resolution mechanical imaging of biological cell and tissue. Recently, several new optical modalities based on stimulated Brillouin scattering have been developed for biomechanical research. As the scattering efficiency of the stimulated process is much higher than its counterpart in the spontaneous process, stimulated Brillouin-based methods have the potential to significantly improve the speed and spectral resolution of existing Brillouin microscopy. Here, we review the ongoing technological advancements of three methods, including continuous wave stimulated Brillouin microscopy, impulsive stimulated Brillouin microscopy, and laser-induced picosecond ultrasonics. We describe the physical principle, the representative instrumentation, and biological application of each method. We further discuss the current limitations as well as the challenges for translating these methods into a visible biomedical instrument for biophysics and mechanobiology.

In vivo corneal elastography: A topical review of challenges and opportunities.

Clinical measurement of corneal biomechanics can aid in the early diagnosis, progression tracking, and treatment evaluation of ocular diseases. Over the past two decades, interdisciplinary collaborations between investigators in optical engineering, analytical biomechanical modeling, and clinical research has expanded our knowledge of corneal biomechanics. These advances have led to innovations in testing methods (ex vivo, and recently, in vivo) across multiple spatial and strain scales. However, in vivo measurement of corneal biomechanics remains a long-standing challenge and is currently an active area of research. Here, we review the existing and emerging approaches for in vivo corneal biomechanics evaluation, which include corneal applanation methods, such as ocular response analyzer (ORA) and corneal visualization Scheimpflug technology (Corvis ST), Brillouin microscopy, and elastography methods, and the emerging field of optical coherence elastography (OCE). We describe the fundamental concepts, analytical methods, and current clinical status for each of these methods. Finally, we discuss open questions for the current state of in vivo biomechanics assessment techniques and requirements for wider use that will further broaden our understanding of corneal biomechanics for the detection and management of ocular diseases, and improve the safety and efficacy of future clinical practice.

Dispersive coherent Brillouin scattering spectroscopy.

Frequency- and time-domain Brillouin scattering spectroscopy are powerful tools to read out the mechanical properties of complex systems in material and life sciences. Indeed, coherent acoustic phonons in the time-domain method offer superior depth resolution and a stronger signal than incoherent acoustic phonons in the frequency-domain method. However, it requires scanning of delay time between laser pulses for pumping and probing coherent acoustic phonons. Here, we present Brillouin scattering spectroscopy that spans the time and frequency domains to allow the multichannel detection of Brillouin scattering light from coherent acoustic phonons. Our technique traces the time-evolve Brillouin oscillations at the instantaneous frequency of a chromatic-dispersed laser pulse. The spectroscopic heterodyning of Brillouin scattering light in the frequency domain allows a single-frame readout of gigahertz-frequency oscillations with a spectrometer. As a proof of concept, we imaged heterogeneous thin films and biological cells over a wide bandwidth with nanometer depth resolution.

In vivo assessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy.

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.

Timothy Grass Pollen Induces Spatial Reorganisation of F-Actin and Loss of Junctional Integrity in Respiratory Cells.

Grass pollens have been identified as mediators of respiratory distress, capable of exacerbating respiratory diseases including epidemic thunderstorm asthma (ETSA). It is hypothesised that during thunderstorms, grass pollen grains swell to absorb atmospheric water, rupture, and release internal protein content to the atmosphere. The inhalation of atmospheric grass pollen proteins results in deadly ETSA events. We sought to identify the underlying cellular mechanisms that may contribute towards the severity of ETSA in temperate climates using Timothy grass (Phleum pratense). Respiratory cells exposed to Timothy grass pollen protein extract (PPE) caused cells to undergo hypoxia ultimately triggering the subcellular re-organisation of F-actin from the peri junctional belt to cytoplasmic fibre assembly traversing the cell body. This change in actin configuration coincided with the spatial reorganisation of microtubules and importantly, decreased cell compressibility specifically at the cell centre. Further to this, we find that the pollen-induced reorganisation of the actin cytoskeleton prompting secretion of the pro-inflammatory cytokine, interleukin-8. In addition, the loss of peri-junctional actin following exposure to pollen proteins was accompanied by the release of epithelial transmembrane protein, E-cadherin from cell-cell junctions resulting in a decrease in epithelial barrier integrity. We demonstrate that Timothy grass pollen regulates F-actin dynamics and E-cadherin localisation in respiratory cells to mediate cell-cell junctional integrity highlighting a possible molecular pathway underpinning ETSA events.

Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity.

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Tumor cell nuclei soften during transendothelial migration.

During cancer metastasis, tumor cells undergo significant deformation in order to traverse through endothelial cell junctions in the walls of blood vessels. As cells pass through narrow gaps, smaller than the nuclear diameter, the spatial configuration of chromatin must change along with the distribution of nuclear enzymes. Nuclear stiffness is an important determinant of the ability of cells to undergo transendothelial migration, yet no studies have been conducted to assess whether tumor cell cytoskeletal or nuclear stiffness changes during this critical process in order to facilitate passage. To address this question, we employed two non-contact methods, Brillouin confocal microscopy (BCM) and confocal reflectance quantitative phase microscopy (QPM), to track the changes in mechanical properties of live, transmigrating tumor cells in an in vitro collagen gel platform. Using these two imaging modalities to study transmigrating MDA-MB-231, A549, and A375 cells, we found that both the cells and their nuclei soften upon extravasation and that the nuclear membranes remain soft for at least 24 h. These new data suggest that tumor cells adjust their mechanical properties in order to facilitate extravasation.

Predicting the Refractive Index of Tissue Models Using Light Scattering Spectroscopy.

In this work, we report the application of Raman microspectroscopy for analysis of the refractive index of a range of tissue phantoms. Using both a custom-developed setup with visible laser source and a commercial microspectrometer with near infrared laser, we measured the Raman spectra of gelatin hydrogels at various concentrations. By building a calibration curve from measured refractometry data and Raman scattering intensity for different vibrational modes of the hydrogel, we were able to predict the refractive indices of the gels from their Raman spectra. This work highlights the importance of a correlative approach through Brillouin-Raman microspectroscopy for the mechano-chemical analysis of biologically relevant samples.

Mapping Tumor Spheroid Mechanics in Dependence of 3D Microenvironment Stiffness and Degradability by Brillouin Microscopy.

Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids' cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids' mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.

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.

Micromechanical imaging of dentin with Brillouin microscopy.

The structure of teeth can be altered by diet, age or diseases such as caries and sclerosis. It is very important to characterize their mechanical properties to predict and understand tooth decay, design restorative dental procedures, and investigate their tribological behavior. However, existing imaging techniques are not well suited to investigating the micromechanics of teeth, in particular at tissue interfaces. Here, we describe a microscope based on Brillouin light scattering (BLS) developed to probe the spectrum of the light scattered from tooth tissues, from which the mechanical properties (sound velocity, viscosity) can be inferred with a priori knowledge of the refractive index. BLS is an inelastic process that uses the scattering of light by acoustic waves in the GHz range. Our microscope thus reveals the mechanical properties at the micrometer scale without contact with the sample. BLS signals show significant differences between sound tissues and pathological lesions, and can be used to precisely delineate carious dentin. We also show maps of the sagittal and transversal planes of sound tubular dentin that reveal its anisotropic microstructure at 1 µm resolution. Our observations indicate that the collagen-based matrix of dentine is the main load-bearing structure, which can be considered as a fiber-reinforced composite. In the vicinity of polymeric tooth-filling materials, we observed the infiltration of the adhesive complex into the opened tubules of sound dentine. The ability to probe the quality of this interfacial layer could lead to innovative designs of biomaterials used for dental restorations in contemporary adhesive dentistry, with possible direct repercussions on decision-making during clinical work. STATEMENT OF SIGNIFICANCE: Mechanical properties of teeth can be altered by diet, age or diseases. Yet existing imaging modalities cannot reveal the micromechanics of the tooth. Here we developed a new type of microscope that uses the scattering of a laser light by naturally-occurring acoustic waves to probe mechanical changes in tooth tissues at a sub-micrometer scale without contact to the sample. We observe significant mechanical differences between healthy tissues and pathological lesions. The contrast in mechanical properties also reveals the microstructure of the polymer-dentin interfaces. We believe that this new development of laser spectroscopy is very important because it should lead to innovative designs of biomaterials used for dental restoration, and allow delineating precisely destructed dentin for minimally-invasive strategies.