Brillouin Imaging Research Articles

Chemistry matters: A side-by-side comparison of two chemically distinct methacryloylated dECM bioresins for vat photopolymerization

Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2–2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5–83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.

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.

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.

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.

Full-field optical spectroscopy at a high spectral resolution using atomic vapors.

Spectral imaging techniques extract spectral information using dispersive elements in combination with optical microscopes. For rapid acquisition, multiplexing spectral information along one dimension of imaged pixels has been demonstrated in hyperspectral imaging, as well as in Raman and Brillouin imaging. Full-field spectroscopy, i.e., multiplexing where imaged pixels are collected in 2D simultaneously while spectral analysis is performed sequentially, can increase spectral imaging speed, but so far has been attained at low spectral resolutions. Here, we extend 2D multiplexing to high spectral resolutions of ∼80 MHz (∼0.0001 nm) using high-throughput spectral discrimination based on atomic transitions.

Brillouin and Raman imaging of domain walls in periodically-poled 5%-MgO:LiNbO3.

Recently, ferroelectric domain walls (DWs) have attracted considerable attention due to their intrinsic topological effects and their huge potential for optoelectronic applications. In contrast, many of the underlying physical properties and phenomena are not well characterized. In this regard, analyzing the vibrational properties, e.g. by Raman spectroscopy, provides direct access to the various local material properties, such as strains, defects or electric fields. While the optical phonon spectra of DWs have been widely investigated in the past, no reports on the acoustic phonon properties of DWs exist. In this work, we present a joint Raman and Brillouin visualization of ferroelectric DWs in the model ferroelectric lithium niobate. This is possible by using a combined Raman and virtually imaged phased array Brillouin setup. Here, we show that DWs can be visualized via frequency shifts observed in the acoustic phonons, as well. The observed contrast then is qualitatively explained by models adapted from Raman spectroscopy. This work, hence, provides a novel route to study ferroelectric DWs and their intrinsic mechanical properties.

Multivariate analysis of Brillouin imaging data by supervised and unsupervised learning.

Brillouin imaging relies on the reliable extraction of subtle spectral information from hyperspectral datasets. To date, the mainstream practice has been to use line fitting of spectral features to retrieve the average peak shift and linewidth parameters. Good results, however, depend heavily on sufficient signal-to-noise ratio and may not be applicable in complex samples that consist of spectral mixtures. In this work, we thus propose the use of various multivariate algorithms that can be used to perform supervised or unsupervised analysis of the hyperspectral data, with which we explore advanced image analysis applications, namely unmixing, classification and segmentation in a phantom and live cells. The resulting images are shown to provide more contrast and detail, and obtained on a timescale ∼102 faster than fitting. The estimated spectral parameters are consistent with those calculated from pure fitting.

Background-free fibre optic Brillouin probe for remote mapping of micromechanics.

Brillouin imaging (BI) has become a valuable tool for micromechanical material characterisation, thanks to extensive progress in instrumentation in the last few decades. This powerful technique is contactless and label-free, thus making it especially suitable for biomedical applications. Nonetheless, to fully harness the non-contact and non-destructive nature of BI, transformational changes in instrumentation are still needed to extend the technology's utility into the domain of in vivo and in situ operation, which we foresee to be particularly crucial for wide spread usage of BI, e.g. in medical diagnostics and pathology screening. This work addresses this challenge by presenting the first demonstration of a fibre-optic Brillouin probe, capable of mapping the micromechanical properties of a tissue-mimicking phantom. This is achieved through combination of miniaturised optical design, advanced hollow-core fibre fabrication and high-resolution 3D printing. Our prototype probe is compact, background-free and possesses the highest collection efficiency to date, thus providing the foundation of a fibre-based Brillouin device for remote, in situ measurements in challenging and otherwise difficult-to-reach environments in biomedical, material science and industrial applications.

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.

On the actual spatial resolution of Brillouin Imaging.

Brillouin imaging is an emerging optical elastography technique that is able to generate maps of the mechanical properties at microscale with great potential in biophysical and biomedical fields. A key parameter is its spatial resolution, which is usually identified with that of the confocal microscope coupled to the Brillouin interferometer. Conversely, here we demonstrate that the mean free path of acoustic phonons plays a major role in defining the resolution, especially for high numerical aperture confocal setups. Surprisingly, the resolution of elastography maps may even deteriorate when decreasing the scattering volume.

Brillouin Microscopy Visualizes Centralized Corneal Edema in Fuchs Endothelial Dystrophy.

To investigate the feasibility of using Brillouin microscopy for assessment of corneal edema in patients with Fuchs endothelial corneal dystrophy (FECD). Brillouin microscopy analyzes the frequency shift of light inelastically scattered by naturally occurring acoustic waves in a small volume of tissue. The resulting frequency shift is a measure of the local hydromechanical properties of the tissue. Participants were scanned using a clinical Brillouin imaging system (780 nm laser, 5 mW), and a color-coded map of the mean Brillouin shift laterally across the corneal stroma was created. Brillouin maps of normal subjects (n = 8) were relatively homogeneous, whereas maps of patients with FECD (n = 7) exhibited significantly reduced Brillouin shifts (unpaired t test, P < 0.001) centrally. The mean difference of 83 MHz corresponds to approximately 3.9% higher water content (percentage difference in volume fraction) in central corneas of the FECD group relative to normal subjects. The Brillouin scan of a patient with FECD 1 month after Descemet membrane endothelial keratoplasty measured a 62 MHz increase in Brillouin shift relative to the preoperative level, indicating normalization of corneal hydration. All patients with FECD scanned exhibited a centralized reduction in Brillouin shift, distinct from the normal subjects measured and consistent with centralized edema characterized by pachymetry. Brillouin scans revealed substantially reduced water content after Descemet membrane endothelial keratoplasty. These results suggest that Brillouin microscopy could aid treatment planning and assessment of FECD. Moreover, corneal hydration mapping may be useful in understanding fluid pump function dynamics of the cornea and developing early interventions for FECD.

Brillouin Light Scattering: Applications in Biomedical Sciences.

Brillouin spectroscopy and imaging are emerging techniques in analytical science, biophotonics, and biomedicine. They are based on Brillouin light scattering from acoustic waves or phonons in the GHz range, providing a nondestructive contactless probe of the mechanics on a microscale. Novel approaches and applications of these techniques to the field of biomedical sciences are discussed, highlighting the theoretical foundations and experimental methods that have been developed to date. Acknowledging that this is a fast moving field, a comprehensive account of the relevant literature is critically assessed here.

Spectral Phasor Analysis for Brillouin Microspectroscopy

Brillouin Light Scattering (BLS) spectroscopy allows for the all-optical measurement of the hypersonic velocities in a sample, from which one can extract high-frequency elastic moduli. Recent advances in high resolution optical imaging spectrometers have made it conducive to studying live biological samples and further its implementation as an imaging modality for the life sciences. One major challenge in this context is the relatively weak BLS signal together with the subtle BLS spectral variations observed in many biological samples. Here we show that using spectral phasor analysis one can more easily distinguish variations in noisy spectra compared to standard least-squares (LS) fitting. There is no fitting in phasor analysis, and it is both robust in regards to unaccounted for functional variations in the spectra, and orders of magnitude faster than LS-fitting. As such it can prove particularly useful for increasing contrast in Brillouin imaging as well as for high-throughput BLS applications such as cell-sorting and medical diagnostics especially for statistically compromised or noisy data.

Detection of proteoglycan loss from articular cartilage using Brillouin microscopy, with applications to osteoarthritis.

The degeneration of articular cartilage (AC) occurs in osteoarthritis (OA), which is a leading cause of pain and disability in middle-aged and older people. The early disease-related changes in cartilage extra-cellular matrix (ECM) start with depletion of proteoglycan (PG), leading to an increase in tissue hydration and permeability. These early compositional changes are small (<10%) and hence difficult to register with conventional non-invasive imaging technologies (magnetic resonance and ultrasound imaging). Here we apply Brillouin microscopy for detecting changes in the mechanical properties and composition of porcine AC. OA-like degradation is mimicked by enzymatic tissue digestion, and we compare Brillouin microscopy measurements against histological staining of PG depletion over varying digestion times and enzyme concentrations. The non-destructive nature of Brillouin imaging technology opens new avenues for creating minimally invasive arthroscopic devices for OA diagnostics and therapeutic monitoring.

Long-term Brillouin imaging of live cells with reduced absorption-mediated damage at 660 nm wavelength.

In Brillouin microscopy, absorption-induced photodamage of incident light is the primary limitation on signal-to-noise ratio in many practical scenarios. Here we show that 660 nm may represent an optimal wavelength for Brillouin microscopy as it offers minimal absorption-mediated photodamage at high Brillouin scattering efficiency and the possibility to use a pure and narrow laser line from solid-state lasing medium. We demonstrate that live cells are ~80 times less susceptible to the 660 nm incident light compared to 532 nm light, which overall allows Brillouin imaging of up to more than 30 times higher SNR. We show that this improvement enables Brillouin imaging of live biological samples with improved accuracy, higher speed and/or larger fields of views with denser sampling.

Mechanical Mapping of Spinal Cord Growth and Repair in Living Zebrafish Larvae by Brillouin Imaging

The mechanical properties of biological tissues are increasingly recognized as important factors in developmental and pathological processes. Most existing mechanical measurement techniques either necessitate destruction of the tissue for access or provide insufficient spatial resolution. Here, we show for the first time to our knowledge a systematic application of confocal Brillouin microscopy to quantitatively map the mechanical properties of spinal cord tissues during biologically relevant processes in a contact-free and nondestructive manner. Living zebrafish larvae were mechanically imaged in all anatomical planes during development and after spinal cord injury. These experiments revealed that Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animal’s natural development. The Brillouin shift within the spinal cord remained comparable during development and transiently decreased during the repair processes after spinal cord transection. By taking into account the refractive index distribution, we explicitly determined the apparent longitudinal modulus and viscosity of different larval zebrafish tissues. Importantly, mechanical properties differed between tissues in situ and in excised slices. The presented work constitutes the first step toward an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a methodical basis to identify key determinants of mechanical tissue properties, and allows us to test their relative importance in combination with biochemical and genetic factors during developmental and regenerative processes.