Brillouin Microscopy Research Articles

Label-Free Techniques for Probing Biomolecular Condensates.

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time. In particular, we review recent years' exciting development of label-free techniques and methodologies. We broadly organize the set of tools into 3 categories: (1) imaging-based techniques, such as transmitted-light microscopy (TLM) and Brillouin microscopy (BM), (2) force spectroscopy techniques, such as atomic force microscopy (AFM) and the optical tweezer (OT), and (3) microfluidic platforms and emerging technologies. We point out the tools' key opportunities, challenges, and future perspectives and analyze their correlative potential as well as compatibility with other techniques. Additionally, we review emerging techniques, namely, differential dynamic microscopy (DDM) and interferometric scattering microscopy (iSCAT), that have huge potential for future applications in studying biomolecular condensates. Finally, we highlight how some of these techniques can be translated for diagnostics and therapy purposes. We hope this Review serves as a useful guide for new researchers in this field and aids in advancing the development of new biophysical tools to study biomolecular condensates.

Brillouin microscopy monitors rapid responses in subcellular compartments

Measurements and imaging of the mechanical response of biological cells are critical for understanding the mechanisms of many diseases, and for fundamental studies of energy, signal and force transduction. The recent emergence of Brillouin microscopy as a powerful non-contact, label-free way to non-invasively and non-destructively assess local viscoelastic properties provides an opportunity to expand the scope of biomechanical research to the sub-cellular level. Brillouin spectroscopy has recently been validated through static measurements of cell viscoelastic properties, however, fast (sub-second) measurements of sub-cellular cytomechanical changes have yet to be reported. In this report, we utilize a custom multimodal spectroscopy system to monitor for the very first time the rapid viscoelastic response of cells and subcellular structures to a short-duration electrical impulse. The cytomechanical response of three subcellular structures - cytoplasm, nucleoplasm, and nucleoli - were monitored, showing distinct mechanical changes despite an identical stimulus. Through this pioneering transformative study, we demonstrate the capability of Brillouin spectroscopy to measure rapid, real-time biomechanical changes within distinct subcellular compartments. Our results support the promising future of Brillouin spectroscopy within the broad scope of cellular biomechanics.

A multi-modal microscope for integrated mapping of cellular forces and Brillouin scattering with high resolution

Mechanical forces and stiffness play key roles in the health and development of cells and tissue, but despite the physical connection between these quantities, they cannot be monitored in parallel in most cases. Here, we introduce a fully integrated microscope that combines a method for high-resolution cell force imaging (elastic resonator interference stress microscopy, ERISM) with non-contact mapping of the elastic properties of cells (via Brillouin microscopy). In order to integrate both techniques, we had to account for the strong back reflection on the surface of the microcavity used for ERISM measurements as well as the local destruction of the cavity under illumination for Brillouin microscopy measurements. Therefore, we developed an elastic optical microcavity with minimal absorption that can perform ERISM measurements without sustaining laser damage during Brillouin microscopy. Furthermore, an unequal-arm Michelson interferometer was designed to suppress the back reflection of the laser on the ERISM microcavity surface using division by amplitude interference to reduce the reflected light and enhance the Brillouin signal. We show the utility of our integrated microscope by simultaneously mapping cellular forces and Brillouin shifts in cultures of fibroblast cells.

Multimode fiber-coupled VIPA spectrometer for high-throughput Brillouin imaging of biological samples

Slow acquisition time and instrument stability are the two major limitations for the application of confocal Brillouin microscopy to biological materials. Although overlooked, coupling the microscope to the spectrometer with a multimode fiber (MMF) is a simple yet viable solution to increase both the detection efficiency and the stability of the classical single-mode fiber-coupled virtually imaged phase array (VIPA) instruments. Here we implement the first successful MMF-coupled VIPA spectrometer for confocal Brillouin applications and present a dimensioning strategy to optimize its collected power. The use of an MMF brings a tremendous improvement on the stability of the spectrometer that allows performing experiments over several weeks without realignment of the device. For instance, we map the Brillouin shift and linewidth in growing ductal and acinar organoids with a spatial resolution of 1×1×6μm3 and 50ms dwell time. Our results clearly reveal the formation of a lumen in these organoids. Careful examination of the data also suggests an increase in the viscosity of the cells of the assembly.

Principal component analysis in application to Brillouin microscopy data

Brillouin microscopy has recently emerged as a new bio-imaging modality that provides information on the microscale mechanical properties of biological materials, cells and tissues. The data collected in a typical Brillouin microscopy experiment represents the high-dimensional set of spectral information, i.e. each pixel within a 2D/3D Brillouin image is associated with hundreds of points of spectral data. Its analysis requires non-trivial approaches due to subtlety in spectral variations as well as spatial and spectral overlaps of measured features. This article offers a guide to the application of Principal Component Analysis (PCA) for processing Brillouin imaging data. Being unsupervised multivariate analysis, PCA is well-suited to tackle processing of complex Brillouin spectra from heterogeneous biological samples with minimal a priori information requirements. We point out the importance of data pre-processing steps in order to improve outcomes of PCA. We also present a strategy where PCA combined with k-means clustering method can provide a working solution to data reconstruction and deeper insights into sample composition, structure and mechanics.

Brillouin microscopic imaging of ex-vivo porcine eye using VIPA-CMOS-based spectrometer

The research on non-invasive ophthalmic imaging has become a hot topic in diagnosing ophthalmic diseases, especially assessment of corneal biomechanics has been an unmet clinical need in ophthalmology for many years. The Brillouin microscopy technique based on spontaneous Brillouin scattering has emerged as a unique elastography method due to its advantages of non-contact, label-free, and high-resolution biomechanical imaging of biological tissues. Here, we developed a three-dimensional (3D) optical microscope system for ex-vivo porcine cornea Brillouin imaging. To do this, we designed and experimentally demonstrated a setup with double-stage VIPA etalons and CMOS camera that integrate to a fully parallel dispersive Brillouin spectrometer. The designed spectrometer exhibits a smaller dispersion rate to enhance the spectral sampling rate and downsizes the size of the double-stage-based system. Using the optical microscope system, we measured and mapped the Brillouin frequency shifts of the cornea and crystalline lens in ex-vivo porcine eyes at micron-scale spatial resolution, and a spectral accuracy of < 50 MHz can be achieved without interpolating the spectra. This work is essential for future applications of Brillouin microscopy in clinical diagnosis and treatment of ophthalmic diseases.

High-speed impulsive stimulated Brillouin microscopy

Brillouin microscopy, which maps the elastic modulus from the frequency shift of scattered light, has evolved to a faster speed for the investigation of rapid biomechanical changes. Impulsive stimulated Brillouin scattering (ISBS) spectroscopy has the potential to speed up measurement through the resonant amplification interaction from pulsed excitation and time-domain continuous detection. However, significant progress has not been achieved due to the limitation in signal-to-noise ratio (SNR) and the corresponding need for excessive averaging to maintain high spectral precision. Moreover, the limited spatial resolution also hinders its application in mechanical imaging. Here, by scrutinizing the SNR model, we design a high-speed ISBS microscope through multi-parameter optimization including phase, reference power, and acquisition time. Leveraging this, with the further assistance of the Matrix Pencil method for data processing, three-dimensional mechanical images are mapped under multiple contrast mechanisms for a millimeter-scale polydimethylsiloxane pattern immersed in methanol, enabling the identification of these two transparent materials without any contact or labeling. Our experimental results demonstrate the capability to maintain high spectral precision and resolution at a sub-millisecond integration time for one pixel. With a two-order improvement in the speed and a tenfold improvement in the spatial resolution over the state-of-the-art systems, this method makes it possible for ISBS microscopes to sensitively investigate rapid mechanical changes in time and space.

Mendelian Randomisation Analysis of Causal Association between Lifestyle, Health Factors, and Keratoconus.

Keratoconus (KC), a leading cause of vision impairment, has an unclear aetiology. This study used Mendelian randomization (MR) to explore the causal links between various factors (smoking, asthma, Down syndrome, inflammatory bowel disease, atopic dermatitis, and serum 25-hydroxyvitamin D levels) and KC. A two-sample MR design, grounded in genome-wide association study (GWAS) summary statistics, was adopted using data from FinnGen, UK Biobank, and other GWAS-related articles. The inverse-variance weighted (IVW) method was employed, complemented by the Wald ratio method for factors with only one single-nucleotide polymorphism (SNP). Sensitivity and stability were assessed through Cochrane's Q test, the MR-Egger intercept test, MR-PRESSO outlier test, and the leave-one-out analysis. The IVW results for the ORA (Ocular Response Analyzer) biomechanical parameters indicated significant associations between tobacco smoking (CH: p < 0.001; CRF: p = 0.009) and inflammatory bowel disease (CH: p = 0.032; CRF: p = 0.001) and corneal biomechanics. The Wald ratio method showed tobacco smoking was associated with a lower risk of KC (p = 0.024). Conversely, asthma (p = 0.009), atopic dermatitis (p = 0.012), inflammatory bowel disease (p = 0.017), and serum 25-hydroxyvitamin D levels (p = 0.039) were associated with a higher risk of KC by IVW, and the same applied to Down syndrome (p = 0.004) using the Wald ratio. These results underscore the role of corneal biomechanics as potential mediators in KC risk, warranting further investigation using Corvis ST and Brillouin microscopy. The findings emphasise the importance of timely screening for specific populations in KC prevention and management.

In vivo measurement of the biomechanical properties of human skin with motion-corrected Brillouin microscopy.

Biomechanical testing of human skin in vivo is important to study the aging process and pathological conditions such as skin cancer. Brillouin microscopy allows the all-optical, non-contact visualization of the mechanical properties of cells and tissues over space. Here, we use the combination of Brillouin microscopy and optical coherence tomography for motion-corrected, depth-resolved biomechanical testing of human skin in vivo. We obtained two peaks in the Brillouin spectra for the epidermis, the first at 7 GHz and the second near 9-10 GHz. The experimentally measured Brillouin frequency shift of the dermis is lower compared to the epidermis and is 6.8 GHz, indicating the lower stiffness of the dermis.

Brillouin microscopy.

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.

Progressive alteration of murine bladder elasticity in actinic cystitis detected by Brillouin microscopy

Bladder mechanical properties are critical for organ function and tissue homeostasis. Therefore, alterations of tissue mechanics are linked to disease onset and progression. This study aims to characterize the tissue elasticity of the murine bladder wall considering its different anatomical components, both in healthy conditions and in actinic cystitis, a state characterized by tissue fibrosis. Here, we exploit Brillouin microscopy, an emerging technique in the mechanobiology field that allows mapping tissue mechanics at the microscale, in non-contact mode and free of labeling. We show that Brillouin imaging of bladder tissues is able to recognize the different anatomical components of the bladder wall, confirmed by histopathological analysis, showing different tissue mechanical properties of the physiological bladder, as well as a significant alteration in the presence of tissue fibrosis. Our results point out the potential use of Brillouin imaging on clinically relevant samples as a complementary technique to histopathological analysis, deciphering complex mechanical alteration of each tissue layer of an organ that strongly relies on mechanical properties to perform its function.

Acoustic transmission across the interface in impulsive stimulated Brillouin microscopy

Brillouin microscopy, an emerging mechanical imaging technology, has made rapid development in recent years. The Brillouin imaging signal is not only determined by acoustic waves in the optical focus volume but also by acoustic waves outside the focus volume. Here, we study how acoustic propagation across the polydimethylsiloxane (PDMS)–ethanol interface affects the heterodyne impulsive stimulated Brillouin scattering (ISBS) signal. When the acoustic direction is perpendicular to the interface, a frequency component corresponding to the PDMS Brillouin shift appears in the signal although the probe focus is in the ethanol. The transmitted wave from PDMS appears when the acoustic wave propagates to the probe focus. To discuss the acoustic contribution of spatial resolution and the acoustic mode propagation distance, it is necessary to consider the acoustic properties of the medium. The influence of transmitted waves on the interference signal is observed in about 100 μm (phonon mean free path in ethanol). Since ISBS analyzes the signal in the time domain, it is possible to distinguish the transmitted wave from the local signal in a single pixel. Different spatial resolutions are obtained by the time-domain method and frequency-domain method. Using mechanical information outside the optical focus volume, ISBS has the potential to image with fewer pixels, which is more flexible and faster than point-by-point scanning.

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.

Subclinical Keratoconus Detection and Characterization Using Motion-Tracking Brillouin Microscopy

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. Prospective cross-sectional study. Thirty eyes from 30 patients were evaluated, including 15 eyes from 15 bilaterally normal patients and 15 eyes with SKC from 15 patients. 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. Discriminative performance based on AUC, sensitivity, and specificity. 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). 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. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Toward peripheral nerve mechanical characterization using Brillouin imaging spectroscopy.

Peripheral nerves are viscoelastic tissues with unique elastic characteristics. Imaging of peripheral nerve elasticity is important in medicine, particularly in the context of nerve injury and repair. Elasticity imaging techniques provide information about the mechanical properties of peripheral nerves, which can be useful in identifying areas of nerve damage or compression, as well as assessing the success of nerve repair procedures. We aim to assess the feasibility of Brillouin microspectroscopy for peripheral nerve imaging of elasticity, with the ultimate goal of developing a new diagnostic tool for peripheral nerve injury in vivo. Viscoelastic properties of the peripheral nerve were evaluated with Brillouin imaging spectroscopy. An external stress exerted on the fixed nerve resulted in a Brillouin shift. Quantification of the shift enabled correlation of the Brillouin parameters with nerve elastic properties. Brillouin microscopy provides sufficient sensitivity to assess viscoelastic properties of peripheral nerves.

Versatile multimodal modality based on Brillouin light scattering and the photoacoustic effect.

Multimodal optical techniques are useful for the comprehensive characterization of material properties. In this work, we developed a new, to the best of our knowledge, multimodal technology that can simultaneously measure a subset of mechanical, optical, and acoustical properties of the sample and is based on the integration of Brillouin (Br) and photoacoustic (PA) microscopy. The proposed technique can acquire co-registered Br and PA signals from the sample. Importantly, using synergistic measurements of the speed of sound and Brillouin shift, the modality offers a new approach to quantifying the optical refractive index, which is a fundamental property of a material and is not accessible by either technique individually. As a proof of concept, we demonstrated the feasibility of integrating the two modalities and acquired the colocalized Br and time-resolved PA signals in a synthetic phantom made out of kerosene and CuSO4 aqueous solution. In addition, we measured the refractive index values of saline solutions and validated the result. Comparison with previously reported data showed a relative error of 0.3%. This further allowed us to directly quantify the longitudinal modulus of the sample with the colocalized Brillouin shift. While the scope of the current work is limited to introducing the combined Br-PA setup for the first time, we envision that this multimodal modality could open a new path for the multi-parametric analysis of material properties.

Pulsed stimulated Brillouin microscopy.

Stimulated Brillouin scattering is an emerging technique for probing the mechanical properties of biological samples. However, the nonlinear process requires high optical intensities to generate sufficient signal-to-noise ratio (SNR). Here, we show that the SNR of stimulated Brillouin scattering can exceed that of spontaneous Brillouin scattering with the same average power levels suitable for biological samples. We verify the theoretical prediction by developing a novel scheme using low duty cycle, nanosecond pulses for the pump and probe. A shot noise-limited SNR over 1000 was measured with a total average power of 10 mW for 2 ms or 50 mW for 200 µs integration on water samples. High-resolution maps of Brillouin frequency shift, linewidth, and gain amplitude from cells in vitro are obtained with a spectral acquisition time of 20 ms. Our results demonstrate the superior SNR of pulsed stimulated Brillouin over spontaneous Brillouin microscopy.

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

ObjectiveDentin, 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. DesignBoth 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. ResultsComparison 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. ConclusionsWe 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.

Spectral resolution enhancement for impulsive stimulated Brillouin spectroscopy by expanding pump beam geometry.

Brillouin microscopy has recently emerged as a powerful tool for mechanical property measurements in biomedical sensing and imaging applications. Impulsive stimulated Brillouin scattering (ISBS) microscopy has been proposed for faster and more accurate measurements, which do not rely on stable narrow-band lasers and thermally-drifting etalon-based spectrometers. However, the spectral resolution of ISBS-based signal has not been significantly explored. In this report, the ISBS spectral profile has been investigated as a function of the pump beam's spatial geometry, and novel methodologies have been developed for accurate spectral assessment. The ISBS linewidth was found to consistently decrease with increasing pump-beam diameter. These findings provide the means for improved spectral resolution measurements and pave the way to broader applications of ISBS microscopy.

Publisher Correction: Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy

Publisher Correction: Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy