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Purpose: To determine the relationship between regional epithelial thickness and corneal toricity and estimate the epithelium's impact on corneal power in normal corneas., Methods: This was a retrospective case-control study evaluating 200 eyes from 200 patients categorized into one of three groups based on corneal toricity obtained with Scheimpflug imaging: (1) 80 non-toric (NT) eyes (< 0.50 diopters [D] in any meridian), (2) 80 with-the-rule (WTR) eyes (⩾ +1.50 D @90 ± 22.5°), and (3) 40 against-the-rule (ATR) eyes (⩾ +1.50 D @180 ± 22.5°). Epithelial thickness maps (9 mm) were generated using anterior segment optical coherence tomography (ASOCT) imaging (Avanti RTVue XR; Optovue, Inc). Mathematical modeling was used to evaluate the epithelium's impact on corneal power., Results: There were no differences in mean curvature between groups (range: 43.2 to 43.7 µm). Average epithelial thickness profile within the central 9-mm zone varied by approximately 2 µm or less laterally but was thinner superiorly than inferiorly in all three groups (52.45 to 53.36 vs 56.01 to 56.48 µm) with no significant differences between groups. There were no differences in any measured epithelial metric at any location nor any deviation in overall epithelial thickness pattern between groups. There were minimal average variations (< 4 µm) across the cornea between central and peripheral values in any meridian. The modeled impact on corneal optics was a net reduction in curvature by approximately 0.30 D with less than 0.10 D of possible variation resulting from differences in regional thickness., Conclusions: Regional epithelial thickness was unrelated to underlying corneal toricity and had minimal impact on corneal power in normal eyes. There was no evidence that normal epithelium significantly masks underlying normal stromal toricity, nor does the epithelium significantly contribute to corneal power under normal conditions. [ J Refract Surg . 2025;41(2):e91-e101.] ., Competing Interests: Disclosure: GS has patents (UMD and MGH) related to Brillouin technology, and has equity in and is a consultant for Intelon Optics. CJR is a consultant for Ziemer Ophthalmic Systems AG and Oculus Optikgeräte GmbH. The remaining authors have disclosed no potential conflicts of interest, financial or otherwise.

Purpose: To assess the safety of acoustic radiation force optical coherence elastography in the crystalline lens in situ., Methods: Acoustic radiation force (ARF) produced by an immersion single-element ultrasound transducer (nominal frequency = 3.5 MHz) was characterized using a needle hydrophone and used for optical coherence elastography (OCE) of the crystalline lens. Preamplified signals at 50, 100, 250, 500, 750, 1000, and 1250 mV peak amplitude were tested on ex vivo porcine eyes (n = 21). Three-dimensional optical coherence tomography (OCT) and confocal microscopy images were acquired before and after ARF exposure to each signal amplitude to determine damage., Results: The acoustic intensity of the ultrasound transducer at 100-mV preamplified peak amplitude input demonstrated a signal-to-noise ratio high enough for tracking elastic wave propagation in the lens and spatial-peak pulse-average (SPPA) intensity of 24.1 W/cm² and mechanical index (MI) of 0.46. The SPPA intensity was lower than the U.S. Food and Drug Administration (FDA) safety limit (28 W/cm2), but the MI was twice the safety limit (0.23). OCT structural and confocal microscopy images showed damage only at levels exceeding 1150 W/cm2 and 3.2 for SPPA intensity and MI, respectively., Conclusions: OCT and confocal microscopy showed that, even when the intensity exceeded FDA recommendations (>100 mV), no noticeable damage was observed. Although a further reduction in acoustic intensity is necessary to meet FDA safety limits, ARF-based elastography shows promise for safe clinical translation in quantitatively characterizing lenticular biomechanical properties., Translational Relevance: This work assessed the safety standards for acoustic radiation force to be used in human lens elastography according to the FDA safety limits.

Brillouin Light Scattering (BLS) spectroscopy is a non-invasive, non-contact, label-free optical technique that can provide information on the mechanical properties of a material on the sub-micron scale. Over the last decade it has seen increased applications in the life sciences, driven by the observed significance of mechanical properties in biological processes, the realization of more sensitive BLS spectrometers and its extension to an imaging modality. As with other spectroscopic techniques, BLS measurements not only detect signals characteristic of the investigated sample, but also of the experimental apparatus, and can be significantly affected by measurement conditions. The aim of this consensus statement is to improve the comparability of BLS studies by providing reporting recommendations for the measured parameters and detailing common artifacts. Given that most BLS studies of biological matter are still at proof-of-concept stages and use different--often self-built--spectrometers, a consensus statement is particularly timely to assure unified advancement.

Purpose: To quantify corneal cross-linking (CXL)-induced stiffening via mechanical testing to estimate the impact of changes in hydration levels (H) and evaluate depth-dependent tissue hydration after CXL., Methods: Eighty-three porcine corneal buttons were divided into three groups: Standard protocol CXL (S-CXL), accelerated CXL (A-CXL), and untreated (nonirradiated riboflavin-only) controls. Samples were hydrated or dehydrated to modulate H and dynamic mechanical analyzer compression tests were performed to measure Young's modulus (E). To extract the solid tissue network modulus, the cornea was modeled as a biphasic material after measuring E at different H. Corneal hydration was correlated with depth-dependent tissue thickness characterized by confocal reflection microscopy (CRM)., Results: Young's modulus increased fourfold after S-CXL (0.72 ± 0.1 MPa) and threefold after A-CXL E (0.53 ± 0.12 MPa) versus controls (0.17 ± 0.045 MPa). However, H decreased from 4.07 ± 0.35 in controls to 2.06 ± 0.2 after S-CXL and 2.79 ± 0.12 after A-CXL. After H modulation and biphasic mechanical modeling, Young's modulus for corneal solid tissue network showed only a 1.8-fold increase after S-CXL (2.25 MPa) and 1.5-fold increase after A-CXL (1.85 MPa) versus controls (1.22 MPa). With CRM, the overall thickness of the corneal tissue was found to linearly correlate to hydration H as expected. No appreciable depth dependence of hydration-induced thickness changes throughout the corneal buttons were observed., Conclusions: Corneal tissue hydration changes significantly impact measured corneal stiffness after CXL using mechanical testing. Not considering H leads to major overestimation of the stiffening effect of the CXL procedure. Depth-dependence of corneal thickness because of changing hydration is strongly dependent on the integrity of the tissue.

Brillouin spectroscopy has become an important tool for mapping the mechanical properties of biological samples. Recently, stimulated Brillouin scattering ( SBS ) measurements have emerged in this field as a promising technology for lower noise and higher speed measurements. However, further improvements are fundamentally limited by constraints on the optical power level that can be used in biological samples, which effectively caps the gain and signal-to-noise ratio ( SNR ) of SBS biological measurements. This limitation is compounded by practical limits on the optical probe power due to detector saturation thresholds. As a result, SBS -based measurements in biological samples have provided minimal improvements (in noise and imaging speed) compared with spontaneous Brillouin microscopy, despite the potential advantages of the nonlinear scattering process. Here, we consider how a SBS spectrometer can circumvent this fundamental trade-off in the low-gain regime by leveraging the polarization dependence of the SBS interaction to effectively filter the signal from the background light via the polarization pulling effect. We present an analytic model of the polarization pulling detection scheme and describe the trade-space unique to Brillouin microscopy applications. We show that an optimized receiver design could provide >25× improvement in SNR compared to a standard SBS receiver in most typical experimental conditions. We then experimentally validate this model using optical fiber as a simplified test bed. With our experimental parameters, we find that the polarization pulling scheme provides 100× higher SNR than a standard SBS receiver, enabling 100× faster measurements in the low-gain regime. Finally, we discuss the potential for this proposed spectrometer design to benefit low-gain spectroscopy applications such as Brillouin microscopy by enabling pixel dwell times as short as 10 μ s., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

We present the use of stimulated Brillouin scattering spectroscopy to achieve rapid measurements of cell biomechanics in a flow cytometer setup. Specifically, our stimulated Brillouin scattering flow cytometry can acquire at a rate of 200 Hz, with a spectral acquisition time of 5 ms, which marks a 10x improvement compared to previous demonstrations of spontaneous Brillouin scattering flow cytometry. We experimentally validate our stimulated Brillouin scattering flow cytometer by measuring cell populations of normal breast epithelial cells and metastatic breast epithelial cancer cells., Competing Interests: None., (© 2024 Optica Publishing Group.)

Purpose: To determine the misclassification rate of the keratoconus percentage (KISA%) index efficacy in eyes with progressive keratoconus., Methods: This was a retrospective case-control study of consecutive patients with confirmed progressive keratoconus and a contemporaneous normal control group with 1.00 diopters or greater regular astigmatism. Scheimpflug imaging (Pentacam HR) was obtained for all patients. KISA% index and inferior-superior (IS) values were obtained from the Pentacam topometric/keratoconus staging map. Receiver operating characteristic curves were generated to determine the area under the receiver operating characteristic curve (AUROC), sensitivity, and specificity values., Results: There were 160 eyes from 160 patients evaluated, including 80 eyes from 80 patients with progressive keratoconus and 80 eyes from 80 control patients. There were 20 eyes (25%) with progressive keratoconus misclassified by the KISA% index, with 16 eyes (20%) of the progressive keratoconus cohort classified as normal (ie, KISA% < 60). There were 4 eyes (5%) with progressive keratoconus that would classify as having "normal topography" using the published criteria for very asymmetric ectasia with normal topography of KISA% less than 60 and IS value less than 1.45. All controls had a KISA% index value of less than 15. The optimal cut-off value to distinguish cohorts was 15.31 (AUROC = 0.972, 93.75% sensitivity). KISA% index values of 60 and 100 achieved low sensitivity (80% and 73.75%, respectively)., Conclusions: The KISA% index misclassified a significant proportion of eyes with progressive keratoconus as normal. Although highly specific for clinical keratoconus, the KISA% index lacks sensitivity, does not effectively discriminate between normal and abnormal topography, and thus should not be used in large data analysis or artificial intelligence-based modeling. [ J Refract Surg . 2024;40(9):e614-e624.] .

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 - β ) 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 - β , a signaling molecule with diverse cellular functions., Approach: The TGF - β receptor I inhibitor, galunisertib, was employed to assess the mechanical changes resulting from dysregulation of TGF - β . Skin scaffold samples, grouped into three categories (control, TGF - β -treated, and TGF - β + 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 - β -treated group compared with the control group. The difference in wave speed between the control and TGF - β + galunisertib with ( p = 0.10 ) and without AP ( p = 0.36 ) was not significant. Moreover, the TGF - β + galunisertib-treated group exhibited lower wave speed without and with AP and reduced Brillouin frequency shift than the TGF - β -treated group without AP, further strengthening the potential role of TGF - β in regulating the mechanical properties of the samples., Conclusions: These findings offer valuable insights into TGF - β -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., (© 2024 The Authors.)

Background: The brain and spinal cord formation is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Environmental or genetic interferences can impair neurulation, resulting in clinically significant birth defects known collectively as neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling., Results: We demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent reduction of ventral neuroepithelial stiffness in a notochord adjacent area at the level of the rhombomere 5. The formation of cranial and paravertebral ganglia is also impaired in these embryos., Conclusions: This study reveals that hypoplastic hindbrain development, identified by abnormal rhombomere morphology and persistent loss of ventral neuroepithelial stiffness, precedes exencephaly in Fuz ablated murine mutants, indicating that the gene Fuz has a critical function sustaining normal neural tube development and neuronal differentiation., (© 2024 American Association for Anatomy.)

Lens biomechanical properties are critical for our eyes to accommodate. While it is well understood that lens mechanical properties change with age, different experimental techniques have been used over the years, with varying results on how the lens modulus changes. In this study, we developed a spatial-varying elasticity model to characterize the overall elastic modulus of the lens and establish its effect on accommodation. First, to validate the model, ex vivo porcine lenses underwent compression testing using biopsy punches of different diameters to change the percentage of nucleus within samples. Importantly, we found that, indeed, changing nucleus/cortex spatial ratio produces dramatic (∼7-fold) increase in overall sample modulus. Comparing the model with human lens spatial ratios, we demonstrate how changing spatial mechanics are more influential than peak modulus changes on overall elastic modulus. Next, in vivo clinical measurements of the spatial-varying lens modulus were used to generate a simplified mechanical-optical model of accommodation. We defined an ellipsoid lens with patient-derived modulus and geometry measurements, and a statics simulation and ray tracing analysis were performed through the deformed and undeformed lens. The resulting accommodation estimates agree with general accommodation expectations., (© 2024 The Author(s). Published by IOP Publishing Ltd.)

Stimulated Brillouin scattering (SBS) microscopy is a nonlinear all-optical imaging method that provides mechanical contrast based on the interaction of laser radiation and acoustical vibrational modes. Featuring high mechanical specificity and sensitivity, three-dimensional sectioning, and practical imaging times, SBS microscopy with (quasi) continuous wave excitation is rapidly advancing as a promising imaging tool for label-free visualization of viscoelastic information of materials and living biological systems. In this article, we introduce the theory of SBS microscopy and review the current state-of-the-art as well as recent innovations, including different approaches to system designs and data analysis. In particular, various performance parameters of SBS microscopy and its applications in the life sciences are described and discussed. Future perspectives for SBS microscopy are also presented., (© 2024 The Author(s). Published by IOP Publishing Ltd.)

Purpose: P300 is a lysine acetyltransferase that plays a significant role in regulating transcription and the nuclear acetylome. While P300 has been shown to be required for the transcription of certain early flow responsive genes, relatively little is known about its role in the endothelial response to hemodynamic fluid stress. Here we sought to define the role of P300 in mechanotransduction of fluid shear stress in the vascular endothelium., Methods: To characterize cellular mechanotransduction and physical properties after perturbation of P300, we performed bulk RNA sequencing, confocal and Brillouin microscopy, and functional assays on HUVEC., Results: Inhibition of P300 in HUVEC triggers a hyper-alignment phenotype, with cells aligning to flow sooner and more uniformly in the presence of the P300 inhibitor A-485 compared to load controls. Bulk transcriptomics revealed differential expression of genes related to the actin cytoskeleton and migration in cells exposed to A-485. Scratch wound and bead sprouting assays demonstrated that treatment with A-485 increased 2D and 3D migration of HUVEC. Closer examination of filamentous actin revealed the presence of a perinuclear actin cap in both P300 knockdown HUVEC and HUVEC treated with A-485. Interrogation of cell mechanical properties via Brillouin microscopy demonstrated that HUVEC treated with A-485 had lower Brillouin shifts in both the cell body and the nucleus, suggesting that P300 inhibition triggers an increase in cellular and nuclear compliance., Conclusions: Together, these results point to a novel role of P300 in modulating endothelial cell mechanics and mechanotransduction of hemodynamic shear stress., Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00805-2., Competing Interests: Conflict of interestThe authors declare no conflicts of interest., (© The Author(s), under exclusive licence to Biomedical Engineering Society 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Recent research has highlighted the growing significance of the mechanical properties of cells and tissues in the proper execution of physiological functions within an organism; alterations to these properties can potentially result in various diseases. These mechanical properties can be assessed using various techniques that vary in spatial and temporal resolutions as well as applications. Due to the wide range of mechanical behaviors exhibited by cells and tissues, a singular mapping technique may be insufficient in capturing their complexity and nuance. Consequently, by utilizing a combination of methods-multimodal mechanical mapping-researchers can achieve a more comprehensive characterization of mechanical properties, encompassing factors such as stiffness, modulus, viscoelasticity, and forces. Furthermore, different mapping techniques can provide complementary information and enable the exploration of spatial and temporal variations to enhance our understanding of cellular dynamics and tissue mechanics. By capitalizing on the unique strengths of each method while mitigating their respective limitations, a more precise and holistic understanding of cellular and tissue mechanics can be obtained. Here, we spotlight Brillouin microscopy (BM) as a noncontact, noninvasive, and label-free mechanical mapping modality to be coutilized alongside established mechanical probing methods. This review summarizes some of the most widely adopted individual mechanical mapping techniques and highlights several recent multimodal approaches demonstrating their utility. We envision that future studies aim to adopt multimodal techniques to drive advancements in the broader realm of mechanobiology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Brillouin spectrometers, used for characterizing material mechanical properties, traditionally employ etalons such as Fabry-Pérot interferometers and virtually imaged phased arrays (VIPA) that use spatial dispersion of the spectrum for measurement. Here, we introduce what we believe to be a novel approach to Brillouin spectroscopy using hot atomic vapors. Using laser induced circular dichroism of the rubidium D2 line in a ladder-type configuration, we developed a narrow-band monochromator for Brillouin analysis. Unlike etalon-based spectrometers, atomic line monochromators operate in free-space, facilitating Brillouin spectroscopy integration with microscopy instruments. We report the transmission and spectral resolution performances of the spectrometer and demonstrate Brillouin spectra measurements in liquids.

During the first month of pregnancy, the brain and spinal cord are formed through a process called neurulation. However, this process can be altered by low serum levels of folic acid, environmental factors, or genetic predispositions. In 2018, a surveillance study in Botswana, a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs, found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result, the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs, we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing, Optical Coherence Tomography (OCT), Optical Coherence Elastography (OCE), and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast, reverberant OCE measurements indicate decreased organoid volumes and internal stiffness., Competing Interests: RF and RC participated in TeratOmic Consulting LLC., a defunct consulting company. RC currently heads DARTox Consulting, LLC. Additionally, RF serves on the editorial board for the Journal Reproductive and Developmental Medicine and receives travel funds to attend editorial board meetings. MS and KL have a financial interest in ElastEye LLC., which is not related to this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Caiaffa, Tukeman, Delgado, Ambekar, Mekonnen, Singh, Rodriguez, Ricco, Kraushaar, Aglyamov, Scarcelli, Larin, Finnell and Cabrera.)

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., Competing Interests: Competing interests M.S. and K.V.L. have a financial interest in ElastEye, which is unrelated to this work. All other authors declare they have no competing interests., (© 2024. Published by The Company of Biologists Ltd.)

Tissues are active materials where epithelial turnover, immune surveillance, and remodeling of stromal cells such as macrophages all regulate form and function. Scattering modalities such as Brillouin microscopy (BM) can non-invasively access mechanical signatures at GHz. However, our traditional understanding of tissue material properties is derived mainly from modalities which probe mechanical properties at different frequencies. Thus, reconciling measurements amongst these modalities remains an active area. Here, we compare optical tweezer active microrheology (OT-AMR) and Brillouin microscopy (BM) to longitudinally map brain development in the larval zebrafish. We determine that each measurement is able to detect a mechanical signature linked to functional units of the brain. We demonstrate that the corrected BM-Longitudinal modulus using a density factor correlates well with OT-AMR storage modulus at lower frequencies. We also show that the brain tissue mechanical properties are dependent on both the neuronal architecture and the presence of macrophages. Moreover, the BM technique is able to delineate the contributions to mechanical properties of the macrophage from that due to colony stimulating factor 1 receptor (CSF1R) mediated stromal remodeling. Here, our data suggest that macrophage remodeling is instrumental in the maintenance of tissue mechanical homeostasis during development. Moreover, the strong agreement between the OT-AM and BM further demonstrates that scattering-based technique is sensitive to both large and minute structural modification in vivo .

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 (K max ), 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, K max , 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., (Copyright © 2023 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Even though the physical nature of shear and longitudinal moduli are different, empirical correlations between them have been reported in several biological systems. This correlation is of fundamental interest and immense practical value in biomedicine due to the importance of the shear modulus and the possibility to map the longitudinal modulus at high-resolution with all-optical spectroscopy. We investigate the origin of such a correlation in hydrogels. We hypothesize that both moduli are influenced in the same direction by underlying physicochemical properties, which leads to the observed material-dependent correlation. Matching theoretical models with experimental data, we quantify the scenarios in which the correlation holds. For polymerized hydrogels, a correlation was found across different hydrogels through a common dependence on the effective polymer volume fraction. For hydrogels swollen to equilibrium, the correlation is valid only within a given hydrogel system, as the moduli are found to have different scalings on the swelling ratio. The observed correlation allows one to extract one modulus from another in relevant scenarios.

The field of Brillouin microscopy and imaging was established approximately 20 years ago, thanks to the development of non-scanning high-resolution optical spectrometers. Since then, the field has experienced rapid expansion, incorporating technologies from telecommunications, astrophotonics, multiplexed microscopy, quantum optics and machine learning. Consequently, these advancements have led to much-needed improvements in imaging speed, spectral resolution and sensitivity. The progress in Brillouin microscopy is driven by a strong demand for label-free and contact-free methods to characterize the mechanical properties of biomaterials at the cellular and subcellular scales. Understanding the local biomechanics of cells and tissues has become crucial in predicting cellular fate and tissue pathogenesis. This Primer aims to provide a comprehensive overview of the methods and applications of Brillouin microscopy. It includes key demonstrations of Brillouin microscopy and imaging that can serve as a reference for the existing research community and new adopters of this technology. The article concludes with an outlook, presenting the authors' vision for future developments in this vibrant field. The Primer also highlights specific examples where Brillouin microscopy can have a transformative impact on biology and biomedicine., Competing Interests: Competing interests The authors declare no competing interests.

Microlasers have been widely used in biosensing applications because of their high sensitivity to changes in local conditions. However, in most applications, the sensitivity limit is not dictated by the microlaser line width but rather by the much worse spectral resolution of the detection system, typically a grating spectrometer. To address this issue, we built and characterized a two-dimensional (2D) dispersion spectrometer with a virtually imaged phase array etalon and a diffraction grating. The spectrometer can analyze microlaser emission with a spectral resolution of better than 0.300 pm, which enables high-precision measurements of spectral shifts in laser peak emission wavelength and sufficient resolution to detect changes in peak line width. Using commercial fluorescent microspheres as the microlasers, the 2D dispersion spectrometer demonstrated a detection limit for the refractive index change of a liquid medium of 1.37 × 10 -5 RIU and a detection limit for absorption changes of less than 0.02 cm -1 ., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Purpose: To characterize focal biomechanical differences between normal, keratoconic, and post-laser vision correction (LVC) corneas using motion-tracking Brillouin microscopy., Design: Prospective cross-sectional study., Methods: Thirty eyes from 30 patients (10 normal controls [Controls], 10 post-LVC, and 10 stage I or II keratoconus [KC]) had Scheimpflug and motion-tracking Brillouin microscopy imaging using a custom-built device. Mean, maximum (max) and minimum (min) Brillouin shift, spatial standard deviation, and max-min values were compared. Min values were correlated with local Brillouin values at multiple Scheimpflug imaging locations., Results: Mean (P < .0003), min (P < .00001), spatial standard deviation (P < .01), and max-min (P < .001) were significantly different between the groups. In post hoc pairwise comparisons, the best differentiators for group comparisons were mean (P = .0004) and min (P = .000002) for Controls vs KC, min (P = .0022) and max-min (P = .002) for Controls vs LVC, and mean (P = .0037) and min (P = .0043) for LVC vs KC. Min (area under the receiver operating characteristic = 1.0) and mean (area under the receiver operating characteristic =  0.96) performed well in differentiating Control and KC eyes. Min values correlated best with Brillouin shift values at the thinnest corneal point (r 2  = 0.871, P = .001) and maximum keratometry value identified in the tangential curvature map (r 2  = 0.840, P = .002)., Conclusions: Motion-tracking Brillouin microscopy effectively characterized focal corneal biomechanical alterations in LVC and KC and clearly differentiated these groups from Controls. Primary motion-tracking Brillouin metrics performed well in differentiating groups as compared with basic Scheimpflug metrics, in contrast to previous Brillouin studies, and identified focal changes after LVC where prior Brillouin studies did not., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)