Confocal Brillouin Microscopy Research Articles

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.

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

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

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.

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.

Droplet-Assisted Microfluidic Fabrication and Characterization of Multifunctional Polysaccharide Microgels Formed by Multicomponent Reactions.

Polysaccharide-based microgels have broad applications in multi-parametric cell cultures, cell-free biotechnology, and drug delivery. Multicomponent reactions like the Passerini three-component and the Ugi four-component reaction are shown in here to be versatile platforms for fabricating these polysaccharide microgels by droplet microfluidics with a narrow size distribution. While conventional microgel formation requires pre-modification of hydrogel building blocks to introduce certain functionality, in multicomponent reactions one building block can be simply exchanged by another to introduce and extend functionality in a library-like fashion. Beyond synthesizing a range of polysaccharide-based microgels utilizing hyaluronic acid, alginate and chitosan, exemplary in-depth analysis of hyaluronic acid-based Ugi four-component gels is conducted by colloidal probe atomic force microscopy, confocal Brillouin microscopy, quantitative phase imaging, and fluorescence correlation spectroscopy to elucidate the capability of microfluidic multicomponent reactions for forming defined polysaccharide microgel networks. Particularly, the impact of crosslinker amount and length is studied. A higher network density leads to higher Young’s moduli accompanied by smaller pore sizes with lower diffusion coefficients of tracer molecules in the highly homogeneous network, and vice versa. Moreover, tailored building blocks allow for crosslinking the microgels and incorporating functional groups at the same time as demonstrated for biotin-functionalized, chitosan-based microgels formed by Ugi four-component reaction. To these microgels, streptavidin-labeled enzymes are easily conjugated as shown for horseradish peroxidase (HRP), which retains its activity inside the microgels.

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.

Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy.

Two-photon polymerization has enabled precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without perturbation of the surrounding non-irradiated regions. 2P-CXL induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.

Quantification of plaque stiffness by Brillouin microscopy in experimental thin cap fibroatheroma.

Plaques vulnerable to rupture are characterized by a thin and stiff fibrous cap overlaying a soft lipid-rich necrotic core. The ability to measure local plaque stiffness directly to quantify plaque stress and predict rupture potential would be very attractive, but no current technology does so. This study seeks to validate the use of Brillouin microscopy to measure the Brillouin frequency shift, which is related to stiffness, within vulnerable plaques. The left carotid artery of an ApoE(-/-)mouse was instrumented with a cuff that induced vulnerable plaque development in nine weeks. Adjacent histological sections from the instrumented and control arteries were stained for either lipids or collagen content, or imaged with confocal Brillouin microscopy. Mean Brillouin frequency shift was 15.79 ± 0.09 GHz in the plaque compared with 16.24 ± 0.15 (p < 0.002) and 17.16 ± 0.56 GHz (p < 0.002) in the media of the diseased and control vessel sections, respectively. In addition, frequency shift exhibited a strong inverse correlation with lipid area of -0.67 ± 0.06 (p < 0.01) and strong direct correlation with collagen area of 0.71 ± 0.15 (p < 0.05). This is the first study, to the best of our knowledge, to apply Brillouin spectroscopy to quantify atherosclerotic plaque stiffness, which motivates combining this technology with intravascular imaging to improve detection of vulnerable plaques in patients.

Brillouin spectroscopy and lens refilling

AbstractThe confocal Brillouin spectroscopy is an innovative measurement method that allows the non‐invasive determination of the rheological properties of materials. Its application in ophthalmology can offer the possibility to determine in‐vivo the biomechanical properties of transparent biological tissue such as the crystalline lens. This seems to be a promising approach concerning current presbyopia research. Due to the spatially resolved detection of the viscoelastic lens properties, a better understanding of the natural aging process of the lens and the influences of different lens opacities on the stiffness is expected. From the obtained spectral data the relative protein levels, the relative refractive index profile and the relative density profile within the lens tissue can be derived. A measurement set‐up for confocal Brillouin microscopy based on spectral analysis of spontaneous Brillouin scattering signals by using a high‐resolution dispersive device is presented. In‐vitro and in‐vivo test results on native and artificial lenses are presented and evaluated concerning their rheological significance.

Mechanical mapping with chemical specificity by confocal Brillouin and Raman microscopy

We describe the first application of confocal Brillouin and Raman microscopy to ex vivo human epithelial tissue - Barrett's oesophagus. This non-invasive label-free approach provides high-resolution mechanical mapping with chemical specificity, opening the route to a new integrated method for multiple biomedical and bioengineering applications, and potentially in vivo real-time diagnostics.

Non-invasive, ortsaufgelöste Bestimmung von Gewebeeigenschaften der Augenlinse hinsichtlich Rheologie, Brechungsindex, Dichte und Proteinkonzentration unter Anwendung der Brillouin-Spektroskopie

The confocal Brillouin spectroscopy is an innovative measurement method that allows the non-invasive determination of the rheological properties of materials. Its application in ophthalmology can offer the possibility to determine in-vivo the deformation properties of sections of transparent biological tissue such as the cornea or eye lens with spatial resolution. This seems to be a promising approach concerning current presbyopia research. Due to the spatially resolved detection of the viscoelastic lens properties, a better understanding of the natural aging process of the lens and the influences of different lens opacities on the stiffness is expected. From the obtained spectral data the relative protein levels, the relative refractive index profile and the relative density profile within the lens tissue can be derived in addition. A measurement set-up for confocal Brillouin microscopy based on spectral analysis of spontaneous Brillouin scattering signals by using a high-resolution dispersive device is presented. First in-vitro test results on animal and human lenses are presented and evaluated concerning their rheological significance. These data are compared with known research results.

Confocal Brillouin microscopy for three-dimensional mechanical imaging

Acoustically induced inelastic light scattering, first reported in 1922 by Brillouin1, allows non-contact, direct readout of the viscoelastic properties of a material and has widely been investigated for material characterization2, structural monitoring3 and environmental sensing4. Extending the Brillouin technique from point sampling spectroscopy to imaging modality5 would open up new possibilities for mechanical imaging, but has been challenging because rapid spectrum acquisition is required. Here, we demonstrate a confocal Brillouin microscope based on a fully parallel spectrometer-a virtually imaged phased array-that improves the detection efficiency by nearly 100-fold over previous approaches. Using the system, we show the first cross-sectional Brillouin imaging based on elastic properties as the contrast mechanism and monitor fast dynamic changes in elastic modulus during polymer crosslinking. Furthermore, we report the first in situ biomechanical measurement of the crystalline lens in a mouse eye. These results suggest multiple applications of Brillouin microscopy in biomedical and biomaterial science.