Nondestructive, quantitative viability analysis of 3D tissue cultures using machine learning image segmentation.

Ascertaining the collective viability of cells in different cell culture conditions has typically relied on averaging colorimetric indicators and is often reported out in simple binary readouts. Recent research has combined viability assessment techniques with image-based deep-learning models to automate the characterization of cellular properties. However, further development of viability measurements to assess the continuity of possible cellular states and responses to perturbation across cell culture conditions is needed. In this work, we demonstrate an image processing algorithm for quantifying features associated with cellular viability in 3D cultures without the need for assay-based indicators. We show that our algorithm performs similarly to a pair of human experts in whole-well images over a range of days and culture matrix compositions. To demonstrate potential utility, we perform a longitudinal study investigating the impact of a known therapeutic on pancreatic cancer spheroids. Using images taken with a high content imaging system, the algorithm successfully tracks viability at the individual spheroid and whole-well level. The method we propose reduces analysis time by 97% in comparison with the experts. Because the method is independent of the microscope or imaging system used, this approach lays the foundation for accelerating progress in and for improving the robustness and reproducibility of 3D culture analysis across biological and clinical research.

Detecting Disruption of HER2 Membrane Protein Organization in Cell Membranes with Nanoscale Precision.

The spatiotemporal organization of proteins within the cell membrane can affect numerous biological functions, including cell signaling, communication, and transportation. Deviations from normal spatial arrangements have been observed in various diseases, and a better understanding of this process is a key stepping stone to advancing development of clinical interventions. However, given the nanometer length scales involved, detecting these subtle changes has primarily relied on complex super-resolution and single-molecule imaging methods. In this work, we demonstrate an alternative fluorescent imaging strategy for detecting protein organization based on a material that exhibits a unique photophysical behavior known as aggregation-induced emission (AIE). Organic AIE molecules have an increase in emission signal when they are in close proximity, and the molecular motion is restricted. This property simultaneously addresses the high background noise and low detection signal that limit conventional widefield fluorescent imaging. To demonstrate the potential of this approach, the fluorescent molecule sensor is conjugated to a human epidermal growth factor receptor 2 (HER2)-specific antibody and used to investigate the spatiotemporal behavior of HER2 clustering in the membrane of HER2-overexpressing breast cancer cells. Notably, the disruption of HER2 clusters in response to an FDA-approved monoclonal antibody therapeutic (Trastuzumab) is successfully detected using a simple widefield fluorescent microscope. While the sensor demonstrated here is optimized for sensing HER2 clustering, it is an easily adaptable platform. Moreover, given the compatibility with widefield imaging, the system has the potential to be used with high-throughput imaging techniques, accelerating investigations into membrane protein spatiotemporal organization.

Dielectric optical waveguide fabricated on a transparent substrate.

Transparent glass substrates are routinely used in the fabrication of metasurfaces, augmented reality (AR), virtual reality (VR), and holographic devices. While readily compatible with photolithographic patterning methods, when electron beam (E-Beam) techniques are used, field distortion and stitching errors can result due to the buildup of charge. A common approach to overcome this issue is to deposit a thin conductive polymer layer (E-Spacer). However, if high-voltage E-Beam is used to achieve nano-features, the polymer conductivity is not sufficient. We have shown that by using chromium (Cr) as an overcoating conductive layer on the resist, we can achieve accurate and seamless patterning in multiple writing fields and used the method to fabricate on-chip Si3N4 waveguides on SiO2. This technique has the potential to enable the fabrication of large-scale integrated photonic systems on transparent or dielectric substrates.

Stretchable optical diffraction grating from poly(acrylic acid)/polyethylene oxide stereocomplex.

Advances in optical materials, which were initially static elements, have enabled dynamically tunable optical diffraction gratings to be designed. One common tuning strategy relies on mechanical deformation of the grating pitch to modify the diffraction pattern. In the present work, we demonstrate an all-polymer tunable diffraction grating fabricated using a modified replica molding process. The poly(acrylic acid) (PAA)/polyethylene oxide (PEO) polymer stereocomplex films exhibit optical transmittance at or above 80% from 500 nm to 1400 nm and stretchability over 800% strain with reversibility under 70% strain. The imprinted gratings are characterized at 633 nm and 1064 nm under a range of strain conditions. The measured tunability agrees with finite element method modeling.

COVID-19 Diagnostics: Past, Present, and Future.

In winter of 2020, SARS-CoV-2 emerged as a global threat, impacting not only health but also financial and political stability. To address the societal need for monitoring the spread of SARS-CoV-2, many existing diagnostic technologies were quickly adapted to detect SARS-CoV-2 RNA and antigens as well as the immune response, and new testing strategies were developed to accelerate time-to-decision. In parallel, the infusion of research support accelerated the development of new spectroscopic methods. While these methods have significantly reduced the impact of SARS-CoV-2 on society when coupled with behavioral changes, they also lay the groundwork for a new generation of platform technologies. With several epidemics on the horizon, such as the rise of antibiotic-resistant bacteria, the ability to quickly pivot the target pathogen of this diagnostic toolset will continue to have an impact.

Engineering photonics solutions for COVID-19.

As the impact of COVID-19 on society became apparent, the engineering and scientific community recognized the need for innovative solutions. Two potential roadmaps emerged: developing short-term solutions to address the immediate needs of the healthcare communities and developing mid/long-term solutions to eliminate the over-arching threat. However, in a truly global effort, researchers from all backgrounds came together in tackling this challenge. Short-term efforts have focused on re-purposing existing technologies and leveraging additive manufacturing techniques to address shortages in personal protective equipment and disinfection. More basic research efforts with mid-term and long-term impact have emphasized developing novel diagnostics and accelerating vaccines. As a foundational technology, photonics has contributed directly and indirectly to all efforts. This perspective will provide an overview of the critical role that the photonics field has played in efforts to combat the immediate COVID-19 pandemic as well as how the photonics community could anticipate contributing to future pandemics of this nature.

Lightweight UV-C disinfection system.

UV-C exposure is an effective disinfectant for a range of bacteria and viruses. As such, UV-C treatment, in combination with a chemical wipe, is a common cleaning protocol in medical facilities. Given the increase in severe bacterial and viral agents in society, having access to environmentally friendly disinfectant methods is of increasing interest. In response, we designed, constructed, and validated a UV-C disinfection system from readily accessible components. To improve the UV-C intensity, the enclosure interior was coated with chrome paint. The system is validated using Bacillus cereus, a gram-positive endospore-forming bacteria.

All-optical reversible control of integrated resonant cavity by a self-assembled azobenzene monolayer.

The next frontier in photonics will rely on the synergistic combination of disparate material systems. One unique organic molecule is azobenzene. This molecule can reversibly change conformations when optically excited in the blue (trans-to-cis) or mid-IR (cis-to-trans). Here, we form an oriented monolayer of azobenzene-containing 4-(4-diethylaminophenylazo)pyridine (Aazo) on SiO2 optical resonators. Due to the uniformity of the Aazo layers, quality factors over 106 are achieved. To control the photo-response, the density of Aazo groups is tuned by integrating methyl spacer molecules. Using a pair of lasers, the molecule is reversibly flipped between molecular conformations, inducing a refractive index change which results in a resonant wavelength shift. The magnitude of the shift scales with the relative surface density of Aazo. To investigate reproducibility and stability of the organic monolayer, three switching cycles are demonstrated, and the performance is consistent even after a device is stored in air for 6 months.

Cascaded Stokes and anti-Stokes laser based on an optical resonator with a self-assembled organic monolayer.

Due to their high circulating intensities, ultra-high quality factor dielectric whispering gallery mode resonators have enabled the development of low threshold Raman microlasers. Subsequently, other Raman-related phenomena, such as cascaded stimulated Raman scattering (CSRS) and stimulated anti-Stokes Raman scattering (SARS), were observed. While low threshold frequency conversion and generation have clear applications, CSRS and SARS have been limited by the low Raman gain. In this work, the surface of a silica resonator is modified with an organic monolayer, increasing the Raman gain. Up to four orders of CSRS are observed with sub-milliwatt (mW) input power, and the SARS efficiency is improved by three orders of magnitude compared to previous studies with hybrid resonators.

Low-tech solutions for the COVID-19 supply chain crisis.

A global effort is ongoing in the scientific community and in the maker movement, which focuses on creating devices and tinkering with them, to reverse-engineer commercial medical equipment and get it to healthcare workers. For these 'low-tech' solutions to have a real impact, it is important for them to coalesce around approved designs.

How to organize an online conference.

The first online-only meeting in photonics, held on 13 January 2020, was a resounding success, with 1100 researchers participating remotely to discuss the latest advances in photonics. Here, the organizers share their tips and advice on how to organize an online conference.

Biomechanical Analysis of Porcine Cartilage Elasticity.

Grafting of tissue-engineered cartilage to joints with osteoarthritis has the potential to supersede arthroplasty as the standard of care. However, in order to support the development of functional tissue engineering methods, the subfailure biomechanics of the individual cartilage types that comprise joints must be determined. Current methods for analyzing tissues are based on imaging and are therefore unable to profile the strain dependence of mechanical behaviors within different cartilage types. Recently, an analysis technique based on Optical Fiber Polarimetric Elastography (OFPE) has overcome these challenges. OFPE has been used to characterize the different mechanical behaviors of a range of unprocessed biomaterials and tissues. In the present work, this technique is used to characterize the biomechanics of both articular cartilage and meniscal fibrocartilage within a porcine knee. OFPE testing of the tissue is conducted over a range of physiological loading and unloading values. These results demonstrate the distinctive mechanics of each cartilage type. Due to their different locations within the knee, each cartilage type exhibits distinctly unique biomechanical behavior. Based on the results of OFPE, we correlate the specific buckling, delamination, and bridging events to maxima and minima along the loading and unloading curves. This provides unprecedented detail with regard to the subfailure biomechanics. This information is integral to the design of the next generation of tissue-engineered constructs. Therefore, OFPE will be used across multiple disciplines to rapidly determine the mechanical behavior of tissue-engineered constructs to support functional tissue engineering efforts.

Raman-Kerr frequency combs in Zr-doped silica hybrid microresonators.

Resonant cavity-enhanced Kerr frequency combs have been demonstrated using a range of cavity materials. Regardless of cavity type, one fundamental challenge is achieving low or flat dispersion while maintaining high-efficiency four-wave mixing (FWM). Here we demonstrate a Raman-Kerr frequency comb using a Zr-doped silica hybrid toroidal microcavity. The Zr-doped layer both flattens the dispersion and increases the stimulated Raman scattering efficiency. This enhancement enables the generation of FWM around both the Stokes and anti-Stokes Raman scattering emissions. As a result, the Raman-Kerr frequency comb spans more than 300 nm in the near-IR region with less than 5.2 mW of input power.

Rapid Diagnostic for Point-of-Care Malaria Screening.

Despite significant success in therapeutic development, malaria remains a widespread and deadly infectious disease in the developing world. Given the nearly 100% efficacy of current malaria therapeutics, the primary barrier to eradication is lack of early diagnosis of the infected population. However, there are multiple strains of malaria. Although significant efforts and resources have been invested in developing antibody-based diagnostic methods for Plasmodium falciparum, a rapid and easy to use screening method capable of detecting all malaria strains has not been realized. Yet, until the entire malaria-infected population receives treatment, the disease will continue to impact society. Here, we report the development of a portable, magneto-optic technology for early stage malaria diagnosis based on the detection of the malaria pigment, hemozoin. Using ß-hematin, a hemozoin mimic, we demonstrate detection limits of <0.0081 µg/mL in 500 µL of whole rabbit blood with no additional reagents required. This level corresponds to <26 parasites/µL, a full order of magnitude below clinical relevance and comparable to or less than existing technologies.

Low-threshold parametric oscillation in organically modified microcavities.

Coherent frequency generators are an enabling platform in basic science and applied technology. Originally reliant on high-power lasers, recently comb generation has been demonstrated in ultrahigh-Q microcavities. The large circulating intensity within the cavity results in strong light-matter interaction, giving rise to Kerr parametric oscillations for comb generation. However, the comb generation threshold is limited by competing nonlinear effects within the cavity material and low intrinsic material Kerr coefficients. We report a new strategy to fabricate near-infrared frequency combs based on combining high-Q microcavities with monomolecular layers of highly nonlinear small molecules. The functionalized microcavities demonstrate high-efficiency parametric oscillation in the near-IR and generate primary frequency combs with 0.88-mW thresholds, improving optical parametric oscillation generation over nonfunctionalized devices by three orders of magnitude. This organic-inorganic approach enables otherwise unattainable performance and will inspire the next generation of integrated photonic device platforms.

Role of extracellular matrix in the biomechanical behavior of pancreatic tissue.

Correlating the biomechanical properties of tissue with its function is an emerging area of research with potential impact in diagnostics, therapeutics, and prognostics. A critical stepping-stone in developing structure-function models is creating methods that can correlate the tissue structure with its mechanical behavior. As an initial step in addressing this challenge, we have characterized the mechanical behavior of unprocessed pancreatic tissue using optical fiber polarimetric elastography. To correlate the observed behavior to physiologically relevant structural features, a series of architectures are designed and fabricated using 3D printing. The mechanical response of the 3D printed elastomeric structures is analyzed using compressive testing and modeled using finite element analysis. The biomechanical behavior and buckling point of the 3D printed structures is used to create a calibration curve to understand the measured response of the resected pancreatic tissue. Based on the modeling and biomimetic results, the biomechanical behavior of pancreatic tissue is likely due to the collagen IV network.

Characterization of the mechanical properties of resected porcine organ tissue using optical fiber photoelastic polarimetry.

Characterizing the mechanical behavior of living tissue presents an interesting challenge because the elasticity varies by eight orders of magnitude, from 50Pa to 5GPa. In the present work, a non-destructive optical fiber photoelastic polarimetry system is used to analyze the mechanical properties of resected samples from porcine liver, kidney, and pancreas. Using a quasi-linear viscoelastic fit, the elastic modulus values of the different organ systems are determined. They are in agreement with previous work. In addition, a histological assessment of compressed and uncompressed tissues confirms that the tissue is not damaged during testing.

High-Speed "4D" Computational Microscopy of Bacterial Surface Motility.

Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven "twitching" motility and flagellum-driven "spinning" and "swarming" motility. Appendage-driven motility is controlled by molecular motors, and analysis of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, we combine electromagnetic field computation and statistical image analysis to generate 3D movies close to a surface at 5 ms time resolution using conventional inverted microscopes. We treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwell's equations and compute the diffracted light intensities associated with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calculations between measured 2D microscopy images and a library of computed light intensities, we demonstrate that near-surface 3D movies of Pseudomonas aeruginosa translational and rotational motion are possible at high temporal resolution. Comparison between computational reconstructions and detailed hydrodynamic calculations reveals that P. aeruginosa act like low Reynolds number spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ∼2 pN µm. Interestingly, our analysis reveals that P. aeruginosa can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface.

Flexible Light-Emitting Nanocomposite Based on ZnO Nanotetrapods.

Flexible, light-emitting materials have shown promise in a wide range of applications. Here, we develop an inverse soft-lithography process for embedding zinc oxide nanotetrapods (ZnO NTP) uniformly and nondestructively into a host matrix. The crystalline NTPs were synthesized using a catalyst-free, environmentally friendly chemical vapor transport method. The fluorescent emission of the ZnO NTPs was measured before and after the embedding process. Cyclical mechanical bend tests (N > 100) were performed. The emission of the nanomaterial remains throughout.

Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids.

Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy.