Brillouin Microscopy Research Articles

Measuring mechanical anisotropy of the cornea with Brillouin microscopy

Load-bearing tissues are typically fortified by networks of protein fibers, often with preferential orientations. This fiber structure imparts the tissues with direction-dependent mechanical properties optimized to support specific external loads. To accurately model and predict tissues’ mechanical response, it is essential to characterize the anisotropy on a microstructural scale. Previously, it has been difficult to measure the mechanical properties of intact tissues noninvasively. Here, we use Brillouin optical microscopy to visualize and quantify the anisotropic mechanical properties of corneal tissues at different length scales. We derive the stiffness tensor for a lamellar network of collagen fibrils and use angle-resolved Brillouin measurements to determine the longitudinal stiffness coefficients (longitudinal moduli) describing the ex vivo porcine cornea as a transverse isotropic material. Lastly, we observe significant mechanical anisotropy of the human cornea in vivo, highlighting the potential for clinical applications of off-axis Brillouin microscopy.

Multimodal imaging system combining optical coherence tomography and Brillouin microscopy for neural tube imaging.

To understand the dynamics of tissue stiffness during neural tube formation and closure in a murine model, we have developed a multimodal, coaligned imaging system combining optical coherence tomography (OCT) and Brillouin microscopy. Brillouin microscopy can map the longitudinal modulus of tissue but cannot provide structural images. Thus, it is limited for imaging dynamic processes such as neural tube formation and closure. To overcome this limitation, we have combined Brillouin microscopy and OCT in one coaligned instrument. OCT provided depth-resolved structural imaging with a micrometer-scale spatial resolution to guide stiffness mapping by Brillouin modality. 2D structural and Brillouin frequency shift maps were acquired of mouse embryos at gestational day (GD) 8.5, 9.5, and 10.5 with the multimodal system. The results demonstrate the capability of the system to obtain structural and stiffness information simultaneously.

Nonlinear microscopy using impulsive stimulated Brillouin scattering for high-speed elastography.

The impulsive stimulated Brillouin microscopy promises fast, non-contact measurements of the elastic properties of biological samples. The used pump-probe approach employs an ultra-short pulse laser and a cw laser to generate Brillouin signals. Modeling of the microscopy technique has already been carried out partially, but not for biomedical applications. The nonlinear relationship between pulse energy and Brillouin signal amplitude is proven with both simulations and experiments. Tayloring of the excitation parameters on the biologically relevant polyacrylamide hydrogels outline sub-ms temporal resolutions at a relative precision of <1%. Brillouin microscopy using the impulsive stimulated scattering therefore exhibits high potential for the measurements of viscoelastic properties of cells and tissues.

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.

Non-Invasive Full Rheological Characterization via Combined Speckle and Brillouin Microscopy

Rheology serves to measure the deformation and flow of materials. Its associated quantities, for example, the Young&#x2019;s modulus, shear modulus, bulk modulus, or longitudinal modulus, are important in the biomedical field, in particular for soft materials, to characterize the response of materials to external force. Usually, mechanical probes, in particular rheometers or atomic force microscopy, are used to characterize these quantities. In the last decade, optical measurements have been derived to obtain these quantities even in small sample volumes. However, usually only one quantity is evaluated using optical techniques, such as Brillouin microscopy, which does not allow a full rheological characterization. The latter requires measuring at least two quantities, which allows for calculating all further rheological properties. In this paper, we aim to close this gap by combining two optical rheology methods, Brillouin microscopy to measure the longitudinal modulus and Laser Speckle Rheology for the shear modulus. We built an optical setup that allows the non-contact and hence non-destructive and non-invasive measurement of both quantities simultaneously in the same sample using a 780 nm, narrow linewidth (&#x007E;50 kHz) laser system. We evaluate our approach using defined samples of glycerol and polydimethylsiloxane and we demonstrate image acquisition using the combined setup. We also investigate porcine corneae, as biological samples, and demonstrate direct measurement of longitudinal modulus and shear modulus and calculation of Young&#x2019;s modulus, bulk modulus, and the Poission ratio, which are all in good agreement with published quantities. In the future, our approach allows for full characterization of the rheology of biological specimens.

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

Simple SummaryLittle is known about how cancer cells adapt their mechanical properties in complex 3D microenvironments. Here we generated different types of tumor spheroids within compliant or stiff hydrogels. We then quantitatively mapped the mechanical properties of these spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiffer hydrogels showed elevated Brillouin shifts, hence spheroids became “stiffer” compared to the ones cultured within compliant gels. The spheroid’s mechanical properties were modulated by various microenvironment properties including matrix stiffness and degradability and the resultant compressive stress but also depending on whether single cells or cell aggregates were analyzed. Moreover, spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels became stiffer and at the same time, less invasive compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and their microenvironment, which is relevant to better understand cancer progression.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.

Mechanical mapping of mammalian follicle development using Brillouin microscopy

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

Heterodyne Brillouin microscopy for biomechanical imaging.

Microscopic variations in material stiffness play a vital role in cellular scale biomechanics, but are difficult to measure in a natural 3D environment. Brillouin microscopy is a promising technology for such applications, providing non-contact label-free measurement of longitudinal modulus at microscopic resolution. Here we develop heterodyne detection to measure Brillouin scattering signals in a confocal microscope setup, providing sensitive detection with excellent frequency resolution and robust operation in the presence of stray light. The functionality of the microscope is characterized and validated, and the imaging capability demonstrated by imaging structure within both a fibrin fiber network and live cells.

Mechanical mapping of bioprinted hydrogel models by brillouin microscopy

Three-dimensional (3D) bioprinting has revolutionised the field of biofabrication by delivering precise, cost-effective and a relatively simple way of engineering in vitro living systems in high volume for use in tissue regeneration, biological modelling, drug testing and cell-based diagnostics. The complexity of modern bioprinted systems requires quality control assessment to ensure the resulting product meets the desired criteria of structural design, micromechanical performance and long-term durability. Brillouin microscopy could be an excellent solution for micromechanical assessment of the bioprinted models during or post-fabrication since this technology is non-destructive, label-free and is capable of microscale 3D imaging. In this work, we demonstrate the application of Brillouin microscopy to 3D imaging of hydrogel microstructures created through drop-on-demand bioprinting. In addition, we show that this technology can resolve variations between mechanical properties of the gels with slightly different polymer fractions. This work confirms that Brillouin microscopy can be seen as a characterisation technology complementary to bioprinting, and in the future can be combined within the printer design to achieve simultaneous real-time fabrication and micromechanical characterisation of in vitro biological systems.

Brillouin Spectroscopy as an Innovative Tool to Investigate Biomechanical Properties of Native Human Aortic Valve and Bioprostheses Tissue

Objective: Proper aortic valve (AV) function is enabled by a complex multi-layered extracellular matrix structure and embedded valvular interstitial and endothelial cells (VICs/VECs). In a highly dynamic environment, VICs and VECs respond to biomechanical stimuli thereby influencing valvular matrix remodelling and pathological changes. Thus, it is of cardinal importance to understand the mechanobiology of AV tissue. It is hypothesized that biomechanical properties of native human AV as well as biological AV prostheses tissue can be determined using Brillouin spectroscopy. Methods: Native AV tissue (n=4) and explanted AV prosthesis (n=1) were obtained from operating theatre after AV replacement, 1mm-thick vibratome cuts were prepared (cross section in radial direction) and directly analysed by means of a confocal Brillouin microscope (10x/0.25NA). Here, the inelastically back-scattered laser light (780nm) is analysed by a custom-build Brillouin spectrometer based on a two-stage virtually imaged phased array (VIPA) set-up. Brillouin maps have been acquired pixel-by-pixel (acquisition time 1s/pixel). Tissue slides adjacent to the vibratome sections were fixed and examined histologically. Results: In stenotic AV tissue mineralized regions in the fibrosa exhibit higher Brillouin shift values compared to non-mineralized spongiosa (Figure 1A). The matrix of the examined explanted AV bioprosthesis (glutaraldehyde-fixed porcine AV) showed higher Brillouin shift values than the massive endocarditic vegetation covering both surfaces of the cusp. Calcified regions at the coaptation area were correlated with higher Brillouin shift values (Figure 1B). Conclusions: Brillouin spectroscopy enables detection of differentiated degrees of tissue stiffness within native stenotic and fibrotic human AV. It represents a promising tool to analyze biomechanical properties of human AV. Figure 1. Procedure of Brillouin spectroscopy on (a) stenotic human aortic valve and (b) aortic valve prothesis (from porcine AV cusps) with florid endocarditis and subsequent histological analysis, high Brillouin shift values indicate enhanced stiffness

Brillouin microscopy for the evaluation of hair micromechanics and effect of bleaching.

Brillouin microscopy is a new form of optical elastography and an emerging technique in mechanobiology and biomedical physics. It was applied here to map the viscoelastic properties of human hair and to determine the effect of bleaching on hair properties. For hair samples, longitudinal measurements (i.e. along the fibre axis) revealed peaks at 18.7 and 20.7GHz at the location of the cuticle and cortex, respectively. For hair treated with a bleaching agent, the frequency shifts for the cuticle and cortex were 19.7 and 21.0GHz, respectively, suggesting that bleaching increases the cuticle modulus and-to a minor extent-the cortex modulus. These results demonstrate the capability of Brillouin spectroscopy to address questions on micromechanical properties of hair and to validate the effect of applied treatments.

Advances in Imaging Technology of Anterior Segment of the Eye.

Advances in imaging technology and computer science have allowed the development of newer assessment of the anterior segment, including Corvis ST, Brillouin microscopy, ultrahigh-resolution optical coherence tomography, and artificial intelligence. They enable accurate and precise assessment of structural and biomechanical alterations associated with anterior segment disorders. This review will focus on these 4 new techniques, and a brief overview of these modalities will be introduced. The authors will also discuss the recent advances in research regarding these techniques and potential application of these techniques in clinical practice. Many studies on these modalities have reported promising results, indicating the potential for more detailed comprehensive understanding of the anterior segment tissues.

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.

Label-free histological imaging of tissues using Brillouin light scattering contrast.

Brillouin light scattering offers a unique label-free approach to measure biomechanical properties non-invasively. While this technique is used in biomechanical analysis of cells and tissues, its potential for visualizing structural features of tissues based on the biomechanical contrast has not been much exploited. Here, we present high-resolution Brillouin microscopy images of four basic tissue types: muscular, connective, epithelial, and nervous tissues. The Brillouin contrast distinguishes between muscle fiber cells and endomysium in skeletal muscle and reveals chondrocytes along with spatially varying stiffness of the extracellular matrix in articular cartilage. The hydration-sensitive contrast can visualize the stratum corneum, epidermis, and dermis in the skin epithelium. In brain tissues, the Brillouin images show the mechanical heterogeneity across the cortex and deeper regions. This work demonstrates the versatility of using the Brillouin shift as histological contrast for examining intact tissue substructures via longitudinal modulus without the need for laborious tissue processing steps.

Mapping mechanical properties of biological materials via an add-on Brillouin module to confocal microscopes.

Several techniques have been developed over the past few decades to assess the mechanical properties of biological samples, which has fueled a rapid growth in the fields of biophysics, bioengineering, and mechanobiology. In this context, Brillouin optical spectroscopy has long been known as an intriguing modality for noncontact material characterization. However, limited by speed and sample damage, it had not translated into a viable imaging modality for biomedically relevant materials. Recently, based on a novel spectroscopy strategy that substantially improves the speed of Brillouin measurement, confocal Brillouin microscopy has emerged as a unique complementary tool to traditional methods as it allows noncontact, nonperturbative, label-free measurements of material mechanical properties. The feasibility and potential of this innovative technique at both the cell and tissue level have been extensively demonstrated over the past decade. As Brillouin technology is rapidly recognized, a standard approach for building and operating Brillouin microscopes is required to facilitate the widespread adoption of this technology. In this protocol, we aim to establish a robust approach for instrumentation, and data acquisition and analysis. By carefully following this protocol, we expect that a Brillouin instrument can be built in 5-9 days by a person with basic optics knowledge and alignment experience; the data acquisition as well as postprocessing can be accomplished within 2-8 h.

Brillouin microscopic depth-dependent analysis of corneal crosslinking performed over or under the LASIK flap.

To determine the impact of corneal crosslinking (CXL) performed over the laser in situ keratomileusis (LASIK) flap using the Standard CXL (S-CXL) protocol or under the flap after flap lift (flap-CXL) on regional corneal stiffness using Brillouin microscopy. University of Southern California Keck School of Medicine, Los Angeles, California, and Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, USA. Laboratory ex vivo experiment. After epithelium debridement, LASIK flaps were created on intact fresh porcine eyes with a mechanical microkeratome. Then, S-CXL (riboflavin applied to the corneal surface followed by 3 mW/cm ultraviolet exposure with the flap in place for 30 minutes) or flap-CXL (riboflavin applied to the stromal bed after reflecting the flap followed by the same ultraviolet A exposure with the flap replaced) was performed. Depth profile of stiffness variation and averaged elastic modulus of anterior, middle, and posterior stroma were determined by analyzing Brillouin maps. Each eye served as its own control. The study was performed on 24 fresh porcine eyes. S-CXL had maximal stiffening impact in the anterior most corneal stroma within the LASIK flap (8.40 ± 0.04 GHz), whereas flap-CXL had lower maximal stiffening impact (8.22 ± 0.03 GHz) (P < .001) that occurred 249 ± 34 μm under the corneal surface. S-CXL increased longitudinal modulus by 6.69% (anterior), 0.48% (middle), and -0.91% (posterior) as compared with flap-CXL, which increased longitudinal modulus by 3.43% (anterior, P < .001), 1.23% (middle, P < .1), and -0.78% (posterior, P = .68). The S-CXL technique generated significantly greater stiffening effect in the anterior cornea than a modified protocol with riboflavin administration under the flap (flap-CXL). Minimal stiffening occurred in the middle or posterior cornea with either protocol.

Divided-aperture confocal Brillouin microscopy for simultaneous high-precision topographic and mechanical mapping.

Confocal Brillouin microscopy (CBM) is a novel and powerful technique for providing non-contact and direct readout of the micro-mechanical properties of a material, and thus used in a broad range of applications, including biological tissue detection, cell imaging, and material characterization in manufacturing. However, conventional CBMs have not enabled high precision mechanical mapping owing to the limited depth of focus and are subject to system drift during long-term measurements. In this paper, a divided-aperture confocal Brillouin microscopy (DCBM) is proposed to improve the axial focusing capability, stability, and extinction ratio of CBM. We exploit high-sensitivity divided-aperture confocal technology to achieve an unprecedented 100-fold enhancement in the axial focusing sensitivity of the existing CBMs, reaching 5 nm, and to enhance system stability. In addition, the dark-field setup improves the extinction ratio by 20 dB. To the best of our knowledge, our method achieves the first in situ topographic imaging and mechanical mapping of the sample and provides a new approach for Brillouin scattering applications in material characterization.

Frequency comb enhanced Brillouin microscopy.

Brillouin light scattering (BLS) microscopy is a well established and powerful technique to study acoustic and magnetic excitations in the frequency domain with sub-micron spatial resolution. Many other spectroscopic techniques have benefited from the introduction of femtosecond laser sources to optically pump and stimulate the sample under investigation. In BLS microscopy, the use of femtosecond lasers as the excitation source introduces several challenges, primarily since the measured frequency shift is small and the signal levels are weak due to the low duty cycle of typical femtosecond lasers. Here we present a method to evade these challenges. A strong enhancement of the weak scattering amplitude on selected modes is observed by pumping the sample with a high repetition rate frequency comb laser source. The laser beam can be focused to the diffraction limit, providing a micron pumping area. We can thus preserve the innate high frequency and spatial resolution of BLS microscopy. Furthermore, we are able to induce a point-like source of mode-selected elementary excitations which propagate away from the pumping spot. We conclude that we have demonstrated frequency comb pumped BLS microscopy as an attractive tool for studies of ultrafast induced laser dynamics directly in the frequency domain.

High-sensitivity and high-specificity biomechanical imaging by stimulated Brillouin scattering microscopy.

Label-free, non-contact imaging with mechanical contrast and optical sectioning is a substantial challenge in microscopy. Spontaneous Brillouin scattering microscopy meets this challenge, but encounters a trade-off between acquisition speed and the specificity for biomechanical constituents with overlapping Brillouin bands. Stimulated Brillouin scattering microscopy overcomes this trade-off and enables the cross-sectional imaging of live Caenorhabditis elegans at the organ and subcellular levels, with both elasticity and viscosity contrasts at high specificity and with practical recording times.

Relevant Length Scales in Brillouin Imaging of Biomaterials: The Interplay between Phonons Propagation and Light Focalization

Recent advances in photonics technologies pushed optical microscopy towards new horizons in materials characterization. In this framework, Brillouin microscopy emerged as an innovative method to provide images of materials with mechanical contrast without any physical contact, but exploiting the light-matter interaction. Brillouin imaging holds a great promise; to allow mechanical analysis inside soft and heterogeneous materials, addressing the pivotal role played by viscoelastic properties in the physiology and pathology of living tissues and cells. Nevertheless, extending the approach of Brillouin imaging to characterize elastic heterogeneities of micro and nanostructured samples is especially challenging, and it poses a critical question about the actual spatial resolution reachable in the mechanical characterization. We focus this critical review on the key quantities that define the spatial resolution in the Brillouin scattering process, and we highlight that not only the optical focalization of the light, but also the acoustic excitations present in the material, influence the information collected from a sample by Brillouin imaging. Referring to the body of knowledge gained in the field of material science, we review new results and recently obtained progresses in the more unexplored context of life science. In future developments, a comprehensive strategy to tackle both the acoustic and optical aspects of the measurement will be required to maximize the efficacy of the technique.