Automated brightfield layerwise evaluation in three-dimensional micropatterning via two-photon polymerization.

Two-photon polymerization (TPP) is an advanced 3D fabrication technique capable of creating features with submicron precision. A primary challenge in TPP lies in the facile and accurate characterization of fabrication quality, particularly for structures possessing complex internal features. In this study, we introduce an automated brightfield layerwise evaluation technique that enables a simple-to-implement approach for in situ monitoring and quality assessment of TPP-fabricated structures. Our approach relies on sequentially acquired brightfield images during the TPP writing process and using background subtraction and image processing to extract layered spatial features. We experimentally validate our method by printing a fibrous tissue scaffold and successfully achieve an overall system-adjusted fidelity of 87.5% in situ. Our method is readily adaptable in most TPP systems and can potentially facilitate high-quality TPP manufacturing of sophisticated microstructures.

Introduction to the special feature: amplify Black voices in optics and photonics.

This multi-journal special issue highlights the work of Black scientists and engineers in optics and photonics to accomplish the goal of engaging the entire optics and photonics community and bring awareness to the quality of their research and contributions to the field.

Shear wave elastography can stratify rectal cancer response to short-course radiation therapy.

Rectal cancer is a deadly disease typically treated using neoadjuvant chemoradiotherapy followed by total mesorectal excision surgery. To reduce the occurrence of mesorectal excision surgery for patients whose tumors regress from the neoadjuvant therapy alone, conventional imaging, such as computed tomography (CT) or magnetic resonance imaging (MRI), is used to assess tumor response to neoadjuvant therapy before surgery. In this work, we hypothesize that shear wave elastography offers valuable insights into tumor response to short-course radiation therapy (SCRT)-information that could help distinguish radiation-responsive from radiation-non-responsive tumors and shed light on changes in the tumor microenvironment that may affect radiation response. To test this hypothesis, we performed elastographic imaging on murine rectal tumors (n = 32) on days 6, 10, 12, 16, 18, 20, 23, and 25 post-tumor cell injection. The study revealed that radiation-responsive and non-radiation-responsive tumors had different mechanical properties. Specifically, radiation-non-responsive tumors showed significantly higher shear wave speed SWS (p < 0.01) than radiation-responsive tumors 11 days after SCRT. Furthermore, there was a significant difference in shear wave attenuation (SWA) (p < 0.01) in radiation-non-responsive tumors 16 days after SCRT compared to SWA measured just one day after SCRT. These results demonstrate the potential of shear wave elastography to provide valuable insights into tumor response to SCRT and aid in exploring the underlying biology that drives tumors' responses to radiation.

Confocal reflectance microscopy for mapping collagen fiber organization in the vitreous gel of the eye.

Vitreous collagen structure plays an important role in ocular mechanics. However, capturing this structure with existing vitreous imaging methods is hindered by the loss of sample position and orientation, low resolution, or a small field of view. The objective of this study was to evaluate confocal reflectance microscopy as a solution to these limitations. Intrinsic reflectance avoids staining, and optical sectioning eliminates the requirement for thin sectioning, minimizing processing for optimal preservation of the natural structure. We developed a sample preparation and imaging strategy using ex vivo grossly sectioned porcine eyes. Imaging revealed a network of uniform diameter crossing fibers (1.1 ± 0.3 µm for a typical image) with generally poor alignment (alignment coefficient = 0.40 ± 0.21 for a typical image). To test the utility of our approach for detecting differences in fiber spatial distribution, we imaged eyes every 1 mm along an anterior-posterior axis originating at the limbus and quantified the number of fibers in each image. Fiber density was higher anteriorly near the vitreous base, regardless of the imaging plane. These data demonstrate that confocal reflectance microscopy addresses the previously unmet need for a robust, micron-scale technique to map features of collagen networks in situ across the vitreous.

Realization of high-dynamic-range broadband magnetic-field sensing with ensemble nitrogen-vacancy centers in diamond.

We present a new magnetometry method integrating an ensemble of nitrogen-vacancy (NV) centers in a single-crystal diamond with an extended dynamic range for monitoring a fast changing magnetic-field. The NV-center spin resonance frequency is tracked using a closed-loop frequency locked technique with fast frequency hopping to achieve a 10 kHz measurement bandwidth, thus allowing for the detection of fast changing magnetic signals up to 0.723 T/s. This technique exhibits an extended dynamic range subjected to the working bandwidth of the microwave source. This extended dynamic range can reach up to 4.3 mT, which is 86 times broader than the intrinsic dynamic range. The essential components for NV spin control and signal processing, such as signal generation, microwave frequency control, data processing, and readout, are integrated in a board-level system. With this platform, we demonstrate a broadband magnetometry with an optimized sensitivity of 4.2 nT Hz-1/2. This magnetometry method has the potential to be implemented in a multichannel frequency locked vector magnetometer suitable for a wide range of practical applications, such as magnetocardiography and high-precision current sensors.

Single-wavelength, single-shot pulse oximetry using an LED-generated vector beam.

Photoplethysmography (PPG) is an optical technique that monitors blood oxygen saturation levels, typically with the use of pulse oximeters. Conventional pulse oximetry estimates the ratio of light absorbed at two wavelengths. Attempts have been made to improve the precision of these measurements by using polarized light, with the tradeoff of requiring multiple sequential measurements. We demonstrate a novel PPG technique that uses radially polarized light generated by a light-emitting diode (LED) to obtain single-shot, blood oxygen-saturation measurements using a single wavelength at a rate of 50 fps. Our work, to the best of our knowledge, presents both a novel use of a vector beam and a first demonstration of vector-beam generation using LEDs.

Excitation of surface plasmon polaritons by diffraction-free and vector beams.

Surface plasmon polaritons (SPPs) are traditionally excited by plane waves within the Rayleigh range of a focused transverse-magnetic (TM) Gaussian beam. Here we investigate and confirm the coupling between SPPs and two-dimensional Gaussian and Bessel-Gauss wave packets, as well as one-dimensional light sheets and space-time wave packets. We encode the incoming wavefronts with spatially varying states of polarization; then we couple the respective TM components of radial and azimuthal vector beam profiles to confirm polarization-correlation and spatial-mode selectivity. Our results do not require material optimization or multi-dimensional confinement via periodically corrugated metal surfaces to achieve coupling at a greater extent, hereby outlining a pivotal, yet commonly overlooked, path towards the development of long-range biosensors and all-optical integrated plasmonic circuits.

Reversible inter-degree-of-freedom optical-coherence conversion via entropy swapping.

The entropy associated with an optical field quantifies the field fluctuations and thus its coherence. Any binary optical degree-of-freedom (DoF) - such as polarization or the field at a pair of points in space - can each carry up to one bit of entropy. We demonstrate here that entropy can be reversibly swapped between different DoFs, such that coherence is converted back and forth between them without loss of energy. Specifically, starting with a spatially coherent but unpolarized field carrying one bit of entropy, we unitarily convert the coherence from the spatial DoF to polarization to produce a spatially incoherent but polarized field by swapping the entropy between the two DoFs. Next, we implement the inverse unitary operator, thus converting the coherence back to yield once again a spatially coherent yet unpolarized field. We exploit the intermediate stage between the two coherence conversions - where the spatial coherence has been converted to the polarization DoF - to verify that the field has become immune to the deleterious impact of spatial phase scrambling. Maximizing the spatial entropy protects the spatial DoF by preventing it from taking on any additional fluctuations. After the second coherence conversion, spatial coherence is readily retrieved, and the effect of spatial phase scrambling circumvented.

Demonstration of speckle resistance using space-time light sheets.

The capacity of self-healing fields to reconstruct after passing through scattering media may prove useful in reducing speckle formation. Here, we study the speckle response of the space-time (ST) light sheet compared to a Gaussian wave packet, Airy beam, and Bessel Gauss beam. We find that the Pearson's correlation coefficient for the ST light sheet is 50%, 48% and 40% larger than that of the Gaussian, Airy beam and Bessel Gauss beams, respectively, demonstrating a strong correlation to an input beam that has not been speckled. These results suggest that the ST light sheet exhibits considerable resistance to speckle generation. We also investigate the speckle response of the ST light sheet at its second-harmonic frequency and observe a mean Pearson's correlation coefficient close to 0.6, comparable to the second-harmonic Bessel Gauss beam, and 2.8 × the value obtained for the second-harmonic Gaussian beam. Our results lend themselves to a variety of applications including bioimaging, communications, and optical tweezers.

Glycosaminoglycans modulate long-range mechanical communication between cells in collagen networks.

Cells can sense and respond to mechanical forces in fibrous extracellular matrices (ECMs) over distances much greater than their size. This phenomenon, termed long-range force transmission, is enabled by the realignment (buckling) of collagen fibers along directions where the forces are tensile (compressive). However, whether other key structural components of the ECM, in particular glycosaminoglycans (GAGs), can affect the efficiency of cellular force transmission remains unclear. Here we developed a theoretical model of force transmission in collagen networks with interpenetrating GAGs, capturing the competition between tension-driven collagen fiber alignment and the swelling pressure induced by GAGs. Using this model, we show that the swelling pressure provided by GAGs increases the stiffness of the collagen network by stretching the fibers in an isotropic manner. We found that the GAG-induced swelling pressure can help collagen fibers resist buckling as the cells exert contractile forces. This mechanism impedes the alignment of collagen fibers and decreases long-range cellular mechanical communication. We experimentally validated the theoretical predictions by comparing the intensity of collagen fiber alignment between cellular spheroids cultured on collagen gels versus collagen­GAG cogels. We found significantly lower intensities of aligned collagen in collagen­GAG cogels, consistent with the prediction that GAGs can prevent collagen fiber alignment. The role of GAGs in modulating force transmission uncovered in this work can be extended to understand pathological processes such as the formation of fibrotic scars and cancer metastasis, where cells communicate in the presence of abnormally high concentrations of GAGs.

Fluid mechanics approach to analyzing collagen fiber organization.

SIGNIFICANCE: The spatial organization of collagen fibers has been used as a biomarker for assessing injury and disease progression. However, quantifying this organization for complex structures is challenging. AIM: To quantify and classify complex collagen fiber organizations. APPROACH: Using quantitative second-harmonic generation (SHG) microscopy, we show that collagen-fiber orientation can be viewed as pseudovector fields. Subsequently, we analyze them using fluid mechanic metrics, such as energy U, enstrophy E, and tortuosity τ. RESULTS: We show that metrics used in fluid mechanics for analyzing fluid flow can be adapted to analyze complex collagen fiber organization. As examples, we consider SHG images of collagenous tissue for straight, wavy, and circular fiber structures. CONCLUSIONS: The results of this study show the utility of the chosen metrics to distinguish diverse and complex collagen organizations. We find that the distribution of values for E and U increases with collagen fiber disorganization, where they divide between low and high values corresponding to uniformly aligned fibers and disorganized collagen fibers, respectively. We also confirm that the values of τ cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present.

Space-time vector light sheets.

We introduce the space-time (ST) vector light sheet. This unique one-dimensional ST wave packet is characterized by classical entanglement (CE), a correlation between at least two non-separable intrinsic degrees-of-freedom (DoFs), which in this case are the spatiotemporal DoFs in parallel with the spatial-polarization DoFs. We experimentally confirm that the ST vector light sheet maintains the intrinsic features of the uniformly polarized ST light sheet, such as near-diffraction-free propagation and self-healing, while also maintaining the intrinsic polarization structure of common vector beams, such as those that are radially polarized and azimuthally polarized. We also show that the vector beam structure of the ST vector light sheet is maintained in the subluminal and superluminal regimes.

Tear propagation in vaginal tissue under inflation.

Vaginal tearing at childbirth is extremely common yet understudied despite the long-term serious consequences on women's health. The mechanisms of vaginal tearing remain unknown, and their knowledge could lead to the development of transformative prevention and treatment techniques for maternal injury. In this study, whole rat vaginas with pre-imposed elliptical tears oriented along the axial direction of the organs were pressurized using a custom-built inflation setup, producing large tear propagation. Large deformations of tears through propagation were analyzed, and nonlinear strains around tears were calculated using the digital image correlation technique. Second harmonic generation microscopy was used to examine collagen fiber organization in mechanically untested and tested vaginal specimens. Tears became increasingly circular under pressure, propagating slowly up to the maximum pressure and then more rapidly. Hoop strains were significantly larger than axial strains and displayed a region- and orientation-dependent response with tear propagation. Imaging revealed initially disorganized collagen fibers that aligned along the axial direction with increasing pressure. Fibers in the near-regions of tear tips aligned toward the hoop direction, hampering tear propagation. Changes in tear geometry, regional strains, and fiber orientation revealed the inherent toughening mechanisms of the vaginal tissue. STATEMENT OF SIGNIFICANCE: Women's reproductive health has historically been understudied despite alarming maternal injury and mortality rates in the world. Maternal injury and disability can be reduced by advancing our limited understanding of the large deformations experienced by women's reproductive organs. This manuscript presents, for the first time, the mechanics of tear propagation in vaginal tissue and changes to the underlying collagen microstructure near to and far from the tear. A novel inflation setup capable of maintaining the in vivo tubular geometry of the vagina while propagating a pre-imposed tear was developed. Toughening mechanisms of the vagina to propagation were examined through measurements of tear geometry, strain distributions, and reorientation of collagen fibers. This research draws from current advances in the engineering science and mechanics fields with the goal of improving maternal health care.

Direct laser writing of volumetric gradient index lenses and waveguides.

Direct laser writing (DLW) has been shown to render 3D polymeric optical components, including lenses, beam expanders, and mirrors, with submicrometer precision. However, these printed structures are limited to the refractive index and dispersive properties of the photopolymer. Here, we present the subsurface controllable refractive index via beam exposure (SCRIBE) method, a lithographic approach that enables the tuning of the refractive index over a range of greater than 0.3 by performing DLW inside photoresist-filled nanoporous silicon and silica scaffolds. Adjusting the laser exposure during printing enables 3D submicron control of the polymer infilling and thus the refractive index and chromatic dispersion. Combining SCRIBE's unprecedented index range and 3D writing accuracy has realized the world's smallest (15 µm diameter) spherical Luneburg lens operating at visible wavelengths. SCRIBE's ability to tune the chromatic dispersion alongside the refractive index was leveraged to render achromatic doublets in a single printing step, eliminating the need for multiple photoresins and writing sequences. SCRIBE also has the potential to form multicomponent optics by cascading optical elements within a scaffold. As a demonstration, stacked focusing structures that generate photonic nanojets were fabricated inside porous silicon. Finally, an all-pass ring resonator was coupled to a subsurface 3D waveguide. The measured quality factor of 4600 at 1550 nm suggests the possibility of compact photonic systems with optical interconnects that traverse multiple planes. SCRIBE is uniquely suited for constructing such photonic integrated circuits due to its ability to integrate multiple optical components, including lenses and waveguides, without additional printed supports.

Quantitative Classification of 3D Collagen Fiber Organization From Volumetric Images.

Collagen fibers in biological tissues have a complex 3D organization containing rich information linked to tissue mechanical properties and are affected by mutations that lead to diseases. Quantitative assessment of this 3D collagen fiber organization could help to develop reliable biomechanical models and understand tissue structure-function relationships, which impact diagnosis and treatment of diseases or injuries. While there are advanced techniques for imaging collagen fibers, published methods for quantifying 3D collagen fiber organization have been sparse and give limited structural information which cannot distinguish a wide range of 3D organizations. In this article, we demonstrate an algorithm for quantitative classification of 3D collagen fiber organization. The algorithm first simulates five groups, or classifications, of fiber organization: unidirectional, crimped, disordered, two-fiber family, and helical. These five groups are widespread in natural tissues and are known to affect the tissue's mechanical properties. We use quantitative metrics based on features such as preferred 3D fiber orientation and spherical variance to differentiate each classification in a repeatable manner. We validate our algorithm by applying it to second-harmonic generation images of collagen fibers in tendon and cervix tissue that has been sectioned in specified orientations, and we find strong agreement between classification from simulated data and the physical fiber organization. Our approach provides insight for interpreting 3D fiber organization directly from volumetric images. This algorithm could be applied to other fiber-like structures that are not necessarily made of collagen.

Harmonic optical tomography of nonlinear structures.

Second-harmonic generation microscopy is a valuable label-free modality for imaging non-centrosymmetric structures and has important biomedical applications from live-cell imaging to cancer diagnosis. Conventional second-harmonic generation microscopy measures intensity signals that originate from tightly focused laser beams, preventing researchers from solving the scattering inverse problem for second-order nonlinear materials. Here, we present harmonic optical tomography (HOT) as a novel modality for imaging microscopic, nonlinear and inhomogeneous objects. The HOT principle of operation relies on inter-ferometrically measuring the complex harmonic field and using a scattering inverse model to reconstruct the three-dimensional distribution of harmonophores. HOT enables strong axial sectioning via the momentum conservation of spatially and temporally broadband fields. We illustrate the HOT operation with experiments and reconstructions on a beta-barium borate crystal and various biological specimens. Although our results involve second-order nonlinear materials, we show that this approach applies to any coherent nonlinear process.

Relaying of the local enhanced electric-field using stacked gold bowtie nanoantennas.

We demonstrate the possibility of designing plasmonic structures that extend, and even further concentrate the field enhancement in the feed-gap region of single-layer Au bowtie nanoantennas (BNAs) hundreds of nanometers away from the initial metal-dielectric interface. The design is based on a stack of Au BNAs with progressively reduced gap distances sandwiched by thin dielectric layers. We find that this stacked BNA geometry also behaves as a near-field focusing lens of nanometer focal length. By merely controlling the number of BNAs in a stack and thickness of the accompanying dielectric layers, we show that the usual fast-damping field enhancement right above a 50 nm thick base BNA can be relayed and maintained at >104 over a distance of at least up to 200 nm above the base BNA surface. Our novel plasmonic structures offer an approach to design plasmonic nanostructures for applications in metamaterial engineering, SERS, and nonlinear optics.

Second-harmonic patterned polarization-analyzed reflection confocal microscopy of stromal collagen in benign and malignant breast tissues.

We present the results of polarimetric analysis of collagen on varying pathologies of breast tissues using second-harmonic patterned polarization-analyzed reflection confocal (SPPARC) microscopy. Experiments are conducted on a breast tissue microarray having benign tissues (BT), malignant invasive lobular carcinoma (ILC), and benign stroma adjacent to the malignant tissues (called the benign adjacent tissue, or BAT). Stroma in BAT and ILC exhibit the largest parameter differences. We observe that stromal collagen readings in ILC show lower depolarization, lower diattenuation and higher linear degree-of-polarization values than stromal collagen in BAT. This suggests that the optical properties of collagen change most in the vicinity of tumors. A similar trend is also exhibited in the non-collagenous extrafibrillar matrix plus cells (EFMC) region. The three highlighted parameters show greatest sensitivity to changes in the polarization response of collagen between pathologies.

Quantitative analysis of the effect of environmental-scanning electron microscopy on collagenous tissues.

Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.