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.

An ultra-small bispecific protein augments tumor penetration and treatment for pancreatic cancer.

PURPOSE: The once highly anticipated antibody-based pathway-targeted therapies have not achieved promising outcomes for deadly pancreatic ductal adenocarcinoma (PDAC), mainly due to drugs' low intrinsic anticancer activity and poor penetration across the dense physiological barrier. This study aims to develop an ultra-small-sized, EGFR/VEGF bispecific therapeutic protein to largely penetrate deep tumor tissue and effectively inhibit PDAC tumor growth in vivo. METHODS: The bispecific protein, Bi-fp50, was constructed by a typical synthetic biology method and labeled with fluorescent dyes for in vitro and in vivo imaging. Physicochemical properties, protein dual-binding affinity, and specificity of the Bi-fp50 were evaluated in several PDAC cell lines. In vitro quantitatively and qualitatively anticancer activity of Bi-fp50 was assessed by live/dead staining, MTT assay, and flow cytometry. In vivo pharmacokinetic and biodistribution were evaluated using blood biopsy samples and near-infrared fluorescence imaging. In vivo real-time tracking of Bi-fp50 in the local tumor was conducted by fibered confocal fluorescence microscopy. The subcutaneous PDAC tumor model was used to assess the in vivo antitumor effect of Bi-fp50. RESULTS: Bi-fp50 with an ultra-small size of 50 kDa (5 ~ 6 nm) showed an excellent binding ability to VEGF and EGFR simultaneously and had enhanced, accumulated binding capability for Bxpc3 PDAC cells compared with anti-VEGF scFv and anti-EGFR scFv alone. Additionally, bi-fp50 significantly inhibited the proliferation and growth of Bxpc3 and Aspc1 PDAC cells even under a relatively low concentration (0.3 µM). It showed synergistically enhanced therapeutic effects relative to two individual scFv and Bi-fp50x control in vitro. The half-life of blood clearance of Bi-fp50 was 4.33 ± 0.23 h. After intravenous injection, Bi-fp50 gradually penetrated the deep tumor, widely distributed throughout the whole tissue, and primarily enriched in the tumor with nearly twice the accumulation than scFv2 in the orthotopic PDAC tumor model. Furthermore, the Bi-fp50 protein could induce broad apoptosis in the whole tumor and significantly inhibited tumor growth 3 weeks after injection in vivo without other noticeable side effects. CONCLUSION: The proof-of-concept study demonstrated that the ultra-small-sized, bispecific protein Bi-fp50 could be a potential tumor suppressor and an efficient, safe theranostic tool for treating PDAC tumors.

Poster Session: Optical coherence elastography for In situ measurement of stiffness increase in posterior sclera after crosslinking.

During eye growth, scleral development critically determine eye size and thus the refractive status of the eye. Scleral remodeling in myopia includes scleral thinning, loss of scleral tissue, and weakening of the mechanical properties. Therefore, an intervention aiming at stiffening scleral tissues (crosslinking, SCXL) may provide a way to prevent or treat myopia. The development of SCXL requires tools to evaluate the effects of crosslinking on the mechanical properties of tissues, particularly in sclera where the mechanical properties are more spatially heterogeneous than in the cornea, anisotropic, and varying locally from the anterior to posterior regions. Here, we apply the high-frequency OCE technique to measure the heterogeneous mechanical properties of posterior scleral tissues and, evaluate the changes in shear moduli after SCXL. As a model system, we use ex vivo in porcine eyes and riboflavin-assisted UV crosslinking. From measured elastic wave speeds (6-16kHz), the average out-of-plane shear modulus was 0.71±0.12MPa (n=20) for normal scleras. After treatment, the shear modulus increased to 1.50±0.39MPa. This 2-fold change was consistent with the increase of static Young's modulus from 5.5±.1 to 9.3±1.9MPa after crosslinking, using conventional uniaxial extensometry. OCE revealed regional stiffness differences across the temporal, nasal, and deeper posterior sclera, demonstrating its potential as a noninvasive tool to test the effect of scleral crosslinking.

Supershear surface waves reveal prestress and anisotropy of soft materials.

Surface waves play important roles in many fundamental and applied areas from seismic detection to material characterizations. Supershear surface waves with propagation speeds greater than bulk shear waves have recently been reported, but their properties are not well understood. Here we describe theoretical and experimental results on supershear surface waves in rubbery materials. We find that supershear surface waves can be supported in viscoelastic materials with no restriction on the shear quality factor. Interestingly, the effect of prestress on the speed of the supershear surface wave is opposite to that of the Rayleigh surface wave. Furthermore, anisotropy of material affects the supershear wave much more strongly than the Rayleigh surface wave. We offer heuristic interpretation as well as theoretical verification of our experimental observations. Our work points to the potential applications of supershear waves for characterizing the bulk mechanical properties of soft solid from the free surface.

Poly(catecholamine) coated CsPbBr3 perovskite microlasers: lasing in water and biofunctionalization.

Lead halide perovskite (LHP) is a promising material for various optoelectronic applications. Surface coating on particles is a common strategy to improve their functionality and environmental stability, but LHP is not amenable to most coating chemistries because of its intrinsic weakness against polar solvents. Here, we describe a novel method of synthesizing LHP microlasers in a super-saturated polar solvent using sonochemistry and applying various functional coatings on individual microlasers in situ. We synthesize cesium lead bromine perovskite (CsPbBr3) microcrystals capped with organic poly-norepinephrine (pNE) layers. The catechol group of pNE coordinates to bromine-deficient lead atoms, forming a defect-passivating and diffusion-blocking shell. The pNE layer enhances the material lifetime of CsPbBr3 in water by 2,000-folds, enabling bright luminescence and lasing from single microcrystals in water. Furthermore, the pNE shell permits biofunctionalization with proteins, small molecules, and lipid bilayers. Luminescence from CsPbBr3 microcrystals is sustained in water over 1 hour and observed in live cells. The functionalization method may enable new applications of LHP laser particles in water-rich environments.

Compact Quantum-Dot Microbeads with Sub-Nanometer Emission Linewidth.

Fluorescent microbeads are widely used for applications in life sciences and medical diagnosis. The spectral contrast and sharpness of photoluminescence are critical in the utilities of microbeads for imaging and multiplexing. Here, we demonstrate microbeads capable of generating single-peak laser emission with a sub-nanometer linewidth. The microbeads are made of quantum dots that are tightly packed and crosslinked via ligand exchange for high optical gain and refractive index as well as material stability. Bright single-mode lasing with no photobleaching is achieved with particle diameters as small as 1.5 µm in the air. Sub-nm lasing emission is maintained even inside high-index surroundings, such as organic solvents and biological tissues. Feasibility of intracellular tagging and multi-color imaging in vivo is demonstrated.

Droplet microfluidic generation of a million optical microparticle barcodes.

Micron-scale barcode particles enable labelling of small objects. Here, we demonstrate high-throughput barcode fabrication inside a microfluidic chip that can embed multiple, dye-doped high quality-factor whispering gallery mode cavities inside aqueous droplets at kilohertz rates. These droplets are then cured to form polyacrylamide hydrogel beads as small as 30 µm in diameter. Optical resonance spectra of the embedded cavities provide the hydrogels with unique barcodes with their diversity combinatorically scaled with the number of embedded cavities. Using 3 cavities per hydrogel, we obtain approximately one million uniquely identifiable, optically readable barcode microparticles.

Targeting CXCR4-dependent immunosuppressive Ly6Clow monocytes improves antiangiogenic therapy in colorectal cancer.

Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6Clow monocytes and not Ly6Chigh monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6Clow monocytes (Cx3cr1-/- mice), Ly6Chigh monocytes (CCR2-/- mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6Clow monocytes or neutrophils improved anti-VEGFR2-induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2-induced tumor growth delay but specific depletion of Ly6G+ neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6Clow monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies.

Measuring mechanical wave speed, dispersion, and viscoelastic modulus of the cornea using optical coherence elastography.

Acoustic wave velocity measurement based on optical coherence tomography (OCT) is a promising approach to assess the mechanical properties of biological tissues and soft materials. While studies to date have demonstrated proof of concept of different ways to excite and detect mechanical waves, the quantitative performance of this modality as mechanical measurement has been underdeveloped. Here, we investigate the frequency dependent measurement of the wave propagation in viscoelastic tissues, using a piezoelectric point-contact probe driven with various waveforms. We found that a frequency range of 2-10 kHz is a good window for corneal elastography, in which the lowest-order flexural waves can be identified in post processing. We tested our system on tissue-simulating phantoms and ex vivo porcine eyes, and demonstrate reproducibility and inter-sample variability. Using the Kelvin-Voigt model of viscoelasticity, we extracted the shear-elastic modulus and viscosity of the cornea and their correlation with the corneal thickness, curvature, and eyeball mass. Our results show that our method can be a quantitative, useful tool for the mechanical analysis of the cornea.

Polyethersulfone optical fibers with thermally induced microbubbles for custom side-scattering profiles.

Polyethersulfone (PES) optical fibers are drawn and thermally processed in order to generate variable side-illumination profiles. The thermal treatment allows microbubbles to be formed in an outer layer of the PES fiber, providing light scattering with controllable amplitudes (0.25-2.5 cm-1). Several fibers with different scattering profiles, such as uniform axial irradiation and multiple irradiation spots, are demonstrated. A small microbubble-induced scattering spot on the surface may be used for side-coupling of ambient light into the fiber. These mechanically flexible all-PES fibers with custom-designable scattering profiles may be useful for spatially tuned delivery of light for various applications, including phototherapy.

Light-Guiding Biomaterials for Biomedical Applications.

Optical techniques used in medical diagnosis, surgery, and therapy require efficient and flexible delivery of light from light sources to target tissues. While this need is currently fulfilled by glass and plastic optical fibers, recent emergence of biointegrated approaches, such as optogenetics and implanted devices, call for novel waveguides with certain biophysical and biocompatible properties and desirable shapes beyond what the conventional optical fibers can offer. To this end, exploratory efforts have begun to harness various transparent biomaterials to develop waveguides that can serve existing applications better and enable new applications in future photomedicine. Here, we review the recent progress in this new area of research for developing biomaterial-based optical waveguides. We begin with a survey of biological light-guiding structures found in plants and animals, a source of inspiration for biomaterial photonics engineering. We describe natural and synthetic polymers and hydrogels that offer appropriate optical properties, biocompatibility, biodegradability, and mechanical flexibility have been exploited for light-guiding applications. Finally, we briefly discuss perspectives on biomedical applications that may benefit from the unique properties and functionalities of light-guiding biomaterials.

Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy.

Current measurements of the biomechanical properties of cells require physical contact with cells or lack subcellular resolution. Here we developed a label-free microscopy technique based on Brillouin light scattering that is capable of measuring an intracellular longitudinal modulus with optical resolution. The 3D Brillouin maps we obtained of cells in 2D and 3D microenvironments revealed mechanical changes due to cytoskeletal modulation and cell-volume regulation.

Brillouin microscopy: assessing ocular tissue biomechanics.

PURPOSE OF REVIEW: Assessment of corneal biomechanics has been an unmet clinical need in ophthalmology for many years. Many researchers and clinicians have identified corneal biomechanics as source of variability in refractive procedures and one of the main factors in keratoconus. However, it has been difficult to accurately characterize corneal biomechanics in patients. The recent development of Brillouin light scattering microscopy heightens the promise of bringing biomechanics into the clinic. The aim of this review is to overview the progress and discuss prospective applications of this new technology. RECENT FINDINGS: Brillouin microscopy uses a low-power near-infrared laser beam to determine longitudinal modulus or mechanical compressibility of tissue by analyzing the return signal spectrum. Human clinical studies have demonstrated significant difference in the elastic properties of normal corneas versus corneas diagnosed with mild and severe keratoconus. Clinical data have also shown biomechanical changes after corneal cross-linking treatment of keratoconus patients. Brillouin measurements of the crystalline lens and sclera have also been demonstrated. SUMMARY: Brillouin microscopy is a promising technology under commercial development at present. The technique enables physicians to characterize the biomechanical properties of ocular tissues.

The influence of hydration on different mechanical moduli of the cornea.

PURPOSE: To determine the interrelation of different elastic moduli of the cornea and to investigate their dependency on corneal hydration. METHODS: Rabbit eyes were divided into four groups. Corneas were excised and mounted into a Barron artificial anterior chamber. Various corneal hydration steady states were achieved with different dextran T-500 concentrations in the anterior chamber, as well as on the corneal anterior surface. The treatment-solutions of each group contained either 5, 10, 15, or 20% w/w dextran. Ultrasound pachymetry was used to measure central corneal thickness. Brillouin microscopy of the central cornea determined the longitudinal bulk modulus by means of Brillouin frequency shift. Subsequently, a 5-mm-wide central strip was taken for extensiometry to measure the tangential elastic modulus. RESULTS: The longitudinal bulk modulus was 1.2-times higher in corneas dehydrated with 20% dextran compared to those hydrated with 5% dextran. In contrast, the tangential elastic modulus increased by 4.4 times. The obtained longitudinal bulk moduli were two orders of magnitude bigger than the tangential elastic moduli. Regression analysis of longitudinal bulk modulus and tangential elastic modulus revealed a quadratic relation. The bulk modulus seemed to be independent of tension, whereas the elastic modulus was tension-dependent. Greater corneal hydration led to significantly thicker pachymetry. CONCLUSION: Corneal biomechanics are highly dependent on the level of corneal hydration. Surprisingly, tangential elastic moduli were more sensitive to hydration changes than longitudinal bulk moduli. A quadratic relation was found between both moduli.

The potential of optofluidic biolasers.

Optofluidic biolasers are emerging as a highly sensitive way to measure changes in biological molecules. Biolasers, which incorporate biological material into the gain medium and contain an optical cavity in a fluidic environment, can use the amplification that occurs during laser generation to quantify tiny changes in biological processes in the gain medium. We describe the principle of the optofluidic biolaser, review recent progress and provide our outlooks on potential applications and directions for developing this technology.

The role of cell body density in ruminant retina mechanics assessed by atomic force and Brillouin microscopy.

Cells in the central nervous system (CNS) respond to the stiffness of their environment. CNS tissue is mechanically highly heterogeneous, thus providing motile cells with region-specific mechanical signals. While CNS mechanics has been measured with a variety of techniques, reported values of tissue stiffness vary greatly, and the morphological structures underlying spatial changes in tissue stiffness remain poorly understood. We here exploited two complementary techniques, contact-based atomic force microscopy and contact-free Brillouin microscopy, to determine the mechanical properties of ruminant retinae, which are built up by different tissue layers. As in all vertebrate retinae, layers of high cell body densities ('nuclear layers') alternate with layers of low cell body densities ('plexiform layers'). Different tissue layers varied significantly in their mechanical properties, with the photoreceptor layer being the stiffest region of the retina, and the inner plexiform layer belonging to the softest regions. As both techniques yielded similar results, our measurements allowed us to calibrate the Brillouin microscopy measurements and convert the Brillouin shift into a quantitative assessment of elastic tissue stiffness with optical resolution. Similar as in the mouse spinal cord and the developing Xenopus brain, we found a strong correlation between nuclear densities and tissue stiffness. Hence, the cellular composition of retinae appears to strongly contribute to local tissue stiffness, and Brillouin microscopy shows a great potential for the application in vivo to measure the mechanical properties of transparent tissues.

The commercialization of genome-editing technologies.

The emergence of new gene-editing technologies is profoundly transforming human therapeutics, agriculture, and industrial biotechnology. Advances in clustered regularly interspaced short palindromic repeats (CRISPR) have created a fertile environment for mass-scale manufacturing of cost-effective products ranging from basic research to translational medicine. In our analyses, we evaluated the patent landscape of gene-editing technologies and found that in comparison to earlier gene-editing techniques, CRISPR has gained significant traction and this has established dominance. Although most of the gene-editing technologies originated from the industry, CRISPR has been pioneered by academic research institutions. The spinout of CRISPR biotechnology companies from academic institutions demonstrates a shift in entrepreneurship strategies that were previously led by the industry. These academic institutions, and their subsequent companies, are competing to generate comprehensive intellectual property portfolios to rapidly commercialize CRISPR products. Our analysis shows that the emergence of CRISPR has resulted in a fivefold increase in genome-editing bioenterprise investment over the last year. This entrepreneurial movement has spurred a global biotechnology revolution in the realization of novel gene-editing technologies. This global shift in bioenterprise will continue to grow as the demand for personalized medicine, genetically modified crops and environmentally sustainable biofuels increases. However, the monopolization of intellectual property, negative public perception of genetic engineering and ambiguous regulatory policies may limit the growth of these market segments.

In vivo fluorescence microscopy: lessons from observing cell behavior in their native environment.

Microscopic imaging techniques to visualize cellular behaviors in their natural environment play a pivotal role in biomedical research. Here, we review how recent technical advances in intravital microscopy have enabled unprecedented access to cellular physiology in various organs of mice in normal and diseased states.

Noninvasive Transdermal Vaccination Using Hyaluronan Nanocarriers and Laser Adjuvant.

Vaccines are commonly administered by injection using needles. Although transdermal microneedles are less-invasive promising alternatives, needle-free topical vaccination without involving physical damage to the natural skin barrier is still sought after as it can further reduce needle-induced anxiety and simply administration. However, this long-standing goal has been elusive since the intact skin is impermeable to most macromolecules. Here, we show an efficient, non-invasive transdermal vaccination in mice by employing two key innovations: first, the use of hyaluronan (HA) as vaccine carriers and, second, non-ablative laser adjuvants. Conjugates of a model vaccine ovalbumin (OVA) and HA-HA-OVA conjugates-induced more effective maturation of dendritic cells in vitro, compared to OVA or HA alone, through synergistic HA receptor-mediated effects. Following topical administration in the back skin, HA-OVA conjugates penetrated into the epidermis and dermis in murine and porcine skins up to 30% of the total applied quantity, as revealed by intravital microscopy and quantitative fluorescence assay. Topical administration of HA-OVA conjugates significantly elevated both anti-OVA IgG antibody levels in serum and IgA antibody levels in bronchioalveolar lavage, with peak levels at 4 weeks, while OVA alone had a negligible effect. An OVA challenge at week 8 elicited strong immune-recall humoral responses. With pre-treatment of the skin using non-ablative fractional laser beams (1410 nm wavelength, 10 ms pulse duration, 0.2 mJ/pulse) as laser adjuvant, strong immunization was achieved with much reduced doses of HA-OVA (1 mg/kg OVA). Our results demonstrate the potential of the non-invasive patch-type transdermal vaccination platform.