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Optical scattering poses a significant challenge to high-resolution microscopy within deep tissue. To accurately predict the performance of various microscopy techniques in thick samples, we present a computational model that efficiently solves Maxwell's equation in highly scattering media. This toolkit simulates the deterioration of the laser beam point spread function (PSF) without making a paraxial approximation, enabling accurate modeling of high-numerical-aperture (NA) objective lenses commonly employed in experiments. Moreover, this framework is applicable to a broad range of scanning microscopy techniques including confocal microscopy, stimulated emission depletion (STED) microscopy, and ground-state depletion microscopy. Notably, the proposed method requires only readily obtainable macroscopic tissue parameters. As a practical demonstration, we investigate the performance of Laguerre-Gaussian (LG) versus Hermite-Gaussian (HG) depletion beams in STED microscopy.
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Microscopia , Microscopia/métodos , Fenômenos Ópticos , Espalhamento de Radiação , Microscopia Confocal/métodosRESUMO
Applications of 3D printing that range from temporary medical devices to environmentally responsible manufacturing would benefit from printable resins that yield polymers with controllable material properties and degradation behavior. Towards this goal, poly(ß-amino ester) (PBAE)-diacrylate resins were investigated due to the wide range of available chemistries and tunable material properties. PBAE-diacrylate resins were synthesized from hydrophilic and hydrophobic chemistries and with varying electron densities on the ester bond to provide control over degradation. Hydrophilic PBAE-diacrylates led to degradation behaviors characteristic of bulk degradation while hydrophobic PBAE-diacrylates led to degradation behaviors dominated initially by surface degradation and then transitioned to bulk degradation. Depending on chemistry, the crosslinked PBAE-polymers exhibited a range of degradation times under accelerated conditions, from complete mass loss in 90 min to minimal mass loss at 45 days. Patterned features with 55 µm resolution were achieved across all resins, but their fidelity was dependent on PBAE-diacrylate molecular weight, reactivity, and printing parameters. In summary, simple chemical modifications in the PBAE-diacrylate resins coupled with projection microstereolithography enables high resolution 3D printed parts with similar architectures and initial properties, but widely different degradation rates and behaviors.
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Volumetric additive manufacturing (VAM) enables rapid printing into a wide range of materials, offering significant advantages over other printing technologies, with a lack of inherent layering of particular note. However, VAM suffers from striations, similar in appearance to layers, and similarly limiting applications due to mechanical and refractive index inhomogeneity, surface roughness, etc. We hypothesize that these striations are caused by a self-written waveguide effect, driven by the gelation material nonlinearity upon which VAM relies, and that they are not a direct recording of non-uniform patterning beams. We demonstrate a simple and effective method of mitigating striations via a uniform optical exposure added to the end of any VAM printing process. We show this step to additionally shorten the period from initial gelation to print completion, mitigating the problem of partially gelled parts sinking before print completion, and expanding the range of resins printable in any VAM printer.
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Poly(ß-amino ester)-diacrylates (PBAE-dAs) are promising resins for three-dimensional (3D) printing. This study investigated the degradation of two PBAEs with different chemistries and kinetic chain lengths. PBAE-dA monomers were synthesized from benzhydrazide and poly(ethylene glycol) (A6) or butanediol (B6) diacrylate and then photopolymerized with pentaerythritol tetrakis(3-mercaptopropionate), which formed thiol-polyacrylate kinetic chains. This tetrathiol acts as a cross-linker and chain-transfer agent that controls the polyacrylate kinetic chain length. A6 networks exhibited bulk degradation, while B6 networks exhibited surface degradation, which transitioned to a combined surface and bulk degradation. Increasing the tetrathiol concentration shortened the polyacrylate kinetic chain and time-to-reverse gelation but degradation mode was unaffected. Hydrolysis occurred primarily through the ß-amino ester. As network hydrophilicity increased, the slower degrading ester in the thiol-polyacrylate chains contributed to degradation. Overall, this work demonstrates control over network degradation rate, mode of degradation, and time-to-reverse gelation in PBAE networks and their application in 3D printing.
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Ésteres , Polímeros , Polietilenoglicóis , Polímeros/farmacologia , Impressão Tridimensional , Compostos de SulfidrilaRESUMO
Understanding the three-dimensional (3D) mechanical and chemical properties of distinctly different, adjacent biological tissues is crucial to mimicking their complex properties with materials. 3D printing is a technique often employed to spatially control the distribution of the biomaterials, such as hydrogels, of interest, but it is difficult to print both mechanically robust (high modulus and toughness) and biocompatible (low modulus) hydrogels in a single structure. Moreover, due to the fast diffusion of mobile species during printing and nonequilibrium swelling conditions of low-solids-content hydrogels, it is challenging to form the high-fidelity structures required to mimic tissues. Here a predictive transport and swelling model is presented to model these effects and then is used to compensate for these effects during printing. This model is validated experimentally by photopatterning spatially distinct hydrogel elastic moduli using a single photo-tunable poly(ethylene glycol) (PEG) pre-polymer solution by sequentially patterning and in-diffusing fresh pre-polymer for further polymerization.
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Transparent substrates introduce challenges in optical metrology, recording, and microscopy. Backside reflections reduce signal to noise, are recorded as artifacts, or introduce spurious signals. These reflections often need to be suppressed, but large angular and spectral bandwidths preclude the use of anti-reflection (AR) coatings. Using elastomeric materials doped with optical absorbers, we detail a method and a materials set for temporary suppression of Fresnel reflections for multiple substrates spanning wide spectral and angular bandwidths. Tuning the refractive index of the elastomer to match a substrate minimizes reflection and the addition of different absorptive dopants allow for either broadband or wavelength-selective reflection suppression. As performance is limited only by the index mismatch, both spectral and angular performances significantly exceed those of AR coatings. We demonstrate reflection suppression in excess of 30 dB spanning a bandwidth over 500 nm. After use, these light traps may be removed and reused without damaging the substrate.
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We show the design and fabrication of high diffraction efficiency, optically recorded gradient-index Fresnel lenses in a two-stage photopolymer. A design analysis reveals that lens f/# is limited by the material refractive index contrast, motivating use of recent high-contrast polymers. The number of pixels required for the optical exposure is typically well beyond available spatial light-modulator resolutions, motivating the use of a photolithographic mask. We use a dithered binary chrome mask with 9000×9000 pixels of 2.5 µm diameter to write lenses up to 23 mm in diameter. Lenses down to f/44 with 76% diffraction efficiency and f/79 with 83% diffraction efficiency are demonstrated.
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Precise direct-write lithography of 3D waveguides or diffractive structures within the volume of a photosensitive material is hindered by the lack of metrology that can yield predictive models for the micron-scale refractive index profile in response to a range of exposure conditions. We apply the transport of intensity equation in conjunction with confocal reflection microscopy to capture the complete spatial frequency spectrum of isolated 10 µm-scale gradient-refractive index structures written by single-photon direct-write laser lithography. The model material, a high-performance two-component photopolymer, is found to be linear, integrating, and described by a single master dose response function. The sharp saturation of this function is used to demonstrate nearly binary, flat-topped waveguide profiles in response to a Gaussian focus.
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We demonstrate that multiple exposures of a two-component holographic photopolymer can quadruple the refractive index contrast of the material beyond the single-exposure saturation limit. Quantitative phase microscopy of isolated structures written by laser direct-write lithography is used to characterize the process. This technique reveals that multiple exposures are made possible by diffusion of the chemical components consumed during writing into the previously exposed regions. The ultimate index contrast is shown to be limited by the solubility of fresh components into the multiply exposed region.
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The high signal-to-noise ratios typical of swept-wavelength interferometry (SWI) enable distance measurements to be superresolved with 2σ uncertainties as low as 10-4-10-5 of Fourier transform-limited resolution. We compare three methods of superresolving SWI distance measurements: Local Linear Regression (LLR), Estimation of Signal Parameters via Rotational Invariance Techniques (ESPRIT), and Nonlinear Least Squares (NLS). We find that the superresolution method limits both measurement precision and minimum superresolvable distance. Measurement uncertainty is determined by both the superresolution method and the SWI hardware, while SWI hardware alone limits the maximum superresolvable distance. For very short distances, between 2 and 20 times the SWI system's Fourier transform-limited resolution, NLS provides unbiased estimates with the least uncertainty. At longer distances, LLR provides the fastest unbiased estimates. LLR and NLS are more noise tolerant than ESPRIT and are found to operate close to the Cramér-Rao bound. With sufficient SNR, they provide 1σ measurement precision of 10-4 of the transform limit.
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We introduce a maskless lithography tool and optically-initiated diffusive photopolymer that enable arbitrary two-dimensional gradient index (GRIN) polymer lens profiles. The lithography tool uses a pulse-width modulated deformable mirror device (DMD) to control the 8-bit gray-scale intensity pattern on the material. The custom polymer responds with a self-developing refractive index profile that is non-linear with optical dose. We show that this nonlinear material response can be corrected with pre-compensation of the intensity pattern to yield high fidelity, optically induced index profiles. The process is demonstrated with quadratic, millimeter aperture GRIN lenses, Zernike polynomials and GRIN Fresnel lenses.
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Volumetric additive manufacturing (VAM) is an emerging layerless method for the rapid processing of reactive resins into 3D structures, where printing is much faster (seconds) than other lithography and direct ink writing methods (minutes to hours). As a vial of resin rotates in the VAM process, patterned light exposure defines a 3D object and then resin that has not undergone gelation can be washed away. Despite the promise of VAM, there are challenges with the printing of soft hydrogel materials from non-viscous precursors, including multi-material constructs. To address this, sacrificial gelatin is used to modulate resin viscosity to support the cytocompatible VAM printing of macromers based on poly(ethylene glycol) (PEG), hyaluronic acid (HA), and polyacrylamide (PA). After printing, gelatin is removed by washing at an elevated temperature. To print multi-material constructs, the gelatin-containing resin is used as a shear-yielding suspension bath (including HA to further modulate bath properties) where ink can be extruded into the bath to define a multi-material resin that can then be processed with VAM into a defined object. Multi-material constructs of methacrylated HA (MeHA) and gelatin methacrylamide (GelMA) are printed (as proof-of-concept) with encapsulated mesenchymal stromal cells (MSCs), where the local hydrogel properties guide cell spreading behavior with culture.
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High refractive index, low birefringence photopolymers were created via the radical-mediated, ring opening homopolymerization of 1,2-dithiolane functionalized monomers and were subsequently evaluated as holographic recording media. This investigation systematically characterized the reaction kinetics, thermodynamics, and volume shrinkage of the 1,2-dithiolane homopolymerization as well as the optical transparency, refractive index, birefringence, and holographic performance of multifunctional 1,2-dithiolane functionalized monomers and their resultant polymers. Real-time kinetic and thermodynamic analyses of a monofunctional 1,2-dithiolane monomer, lipoic acid methyl ester (LipOMe), indicated rapid monomer conversion, exceeding 90% in 60 s, with an overall enthalpy of reaction of 18 ± 1 kJ/mol. The ring-opening polymerization resulted in low shrinkage (10.6 ± 0.3 cm3/mol dithiolane) and a significant bulk refractive index increase (0.030 ± 0.003). The resulting photopolymers exhibited high optical transparency, minimal haze, and negligible birefringence, suggesting the potential of 1,2-homopolymers as optical materials. To further explore the specific capabilities for use as high-performance holographic recording applications, several multifunctional monomers were synthesized with the ethanedithiol lipoic acid monomer (EDT-Lip2) selected for experimentation. Holographic diffraction gratings written using this monomer achieved a peak-to-mean refractive index modulation of 0.008 with minimal haze and birefringence.
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Tissue engineered scaffolds are needed to support physiological loads and emulate the micrometer-scale strain gradients within tissues that guide cell mechanobiological responses. We designed and fabricated micro-truss structures to possess spatially varying geometry and controlled stiffness gradients. Using a custom projection microstereolithography (µSLA) system, using digital light projection (DLP), and photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) hydrogel monomers, three designs with feature sizes < 200 µm were formed: (1) uniform structure with 1 MPa structural modulus ( E ) designed to match equilibrium modulus of healthy articular cartilage, (2) E = 1 MPa gradient structure designed to vary strain with depth, and (3) osteochondral bilayer with distinct cartilage ( E = 1 MPa) and bone ( E = 7 MPa) layers. Finite element models (FEM) guided design and predicted the local mechanical environment. Empty trusses and poly(ethylene glycol) norbornene hydrogel-infilled composite trusses were compressed during X-ray microscopy (XRM) imaging to evaluate regional stiffnesses. Our designs achieved target moduli for cartilage and bone while maintaining 68-81% porosity. Combined XRM imaging and compression of empty and hydrogel-infilled micro-truss structures revealed regional stiffnesses that were accurately predicted by FEM. In the infilling hydrogel, FEM demonstrated the stress-shielding effect of reinforcing structures while predicting strain distributions. Composite scaffolds made from stiff µSLA-printed polymers support physiological load levels and enable controlled mechanical property gradients which may improve in vivo outcomes for osteochondral defect tissue regeneration. Advanced 3D imaging and FE analysis provide insights into the local mechanical environment surrounding cells in composite scaffolds.
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Cartilagem Articular , Hidrogéis , Polietilenoglicóis , Alicerces Teciduais , Hidrogéis/química , Polietilenoglicóis/química , Cartilagem Articular/fisiologia , Cartilagem Articular/diagnóstico por imagem , Animais , Engenharia Tecidual , Análise de Elementos Finitos , Impressão TridimensionalRESUMO
We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform since their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm was enabled by a lithium acylphosphinate (LAP) salt photoinitiator. These wavelengths, in addition to brightfield imaging with a white light LED, could be simultaneously focused by a single oil immersion objective. Large lateral dimensions of the patterned gel/cell structure are achieved by x and y step-and-repeat process. Large thickness is achieved through multi-layer stereolithography, allowing fabrication of precisely-arranged 3D live cell scaffolds with micron-scale structure and millimeter dimensions. Cells are shown to retain viability after the trapping and encapsulation procedure.
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Imageamento Tridimensional/instrumentação , Micromanipulação/métodos , Mioblastos/citologia , Mioblastos/fisiologia , Dispositivos Ópticos , Animais , Linhagem Celular , Desenho de Equipamento , Análise de Falha de Equipamento , CamundongosRESUMO
Photoinhibited superresolution (PInSR) lithography is a two-color, one-photon scheme that promises high throughput far-field patterning at deep subwavelength scales. The technique requires that active species are confined for some minimum time to the illuminated area where they are generated. We investigate here the extent to which this condition is met for published materials. Using spatial and temporal control of focused beams as well as fluorescence recovery after photobleaching (FRAP), we probe the dynamics of photoinhibition in the PInSR material system. Our results indicate fast out-diffusion of unreacted photoinhibitor from the submicron optical spot during the polymerization interval, resulting in uniform rather than structured inhibition. Published results are consistent with this mechanism, indicating that superresolved polymer confinement with PInSR has not yet been shown with structured inhibition. To address the issue, we propose modifications to the material and exposure to slow inhibitor out-diffusion and accelerate polymer gelation.
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Volumetric additive manufacturing is a novel fabrication method allowing rapid, freeform, layer-less 3D printing. Analogous to computer tomography (CT), the method projects dynamic light patterns into a rotating vat of photosensitive resin. These light patterns build up a three-dimensional energy dose within the photosensitive resin, solidifying the volume of the desired object within seconds. Departing from established sequential fabrication methods like stereolithography or digital light printing, volumetric additive manufacturing offers new opportunities for the materials that can be used for printing. These include viscous acrylates and elastomers, epoxies (and orthogonal epoxy-acrylate formulations with spatially controlled stiffness) formulations, tunable stiffness thiol-enes and shape memory foams, polymer derived ceramics, silica-nanocomposite based glass, and gelatin-based hydrogels for cell-laden biofabrication. Here we review these materials, highlight the challenges to adapt them to volumetric additive manufacturing, and discuss the perspectives they present. Supplementary Information: The online version contains supplementary material available at10.1557/s43579-023-00447-x.
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We demonstrate single-mode uniform and parabolically tapered three-dimensional waveguides fabricated via direct-write lithography in diffusion-based photopolymers. Modulation of the writing power is shown to compensate Beer-Lambert absorption in the single-photon initiator and to provide precise control of modal tapers. A laminated sample preparation is introduced to enable full 3D characterization of these modal tapers without the need for sample polishing which is difficult for this class of polymer. The accuracy and repeatability of this modal characterization is shown to allow precise measurement of propagation loss from single samples. These testing procedures are used to demonstrate single-mode waveguides with 0.147 dB/cm excess propagation loss and symmetrical tapers up to 1:2.5 using 1.5 microwatts of continuous write power.
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Modelos Teóricos , Polímeros/química , Refratometria/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Absorção , Simulação por Computador , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Espalhamento de RadiaçãoRESUMO
Growth plate injuries affecting the pediatric population may cause unwanted bony repair tissue that leads to abnormal bone elongation. Clinical treatment involves bony bar resection and implantation of an interpositional material, but success is limited and the bony bar often reforms. No treatment attempts to regenerate the growth plate cartilage. Herein we develop a 3D printed growth plate mimetic composite as a potential regenerative medicine approach with the goal of preventing limb length discrepancies and inducing cartilage regeneration. A poly(ethylene glycol)-based resin was used with digital light processing to 3D print a mechanical support structure infilled with a soft cartilage-mimetic hydrogel containing chondrogenic cues. Our biomimetic composite has similar mechanical properties to native rabbit growth plate and induced chondrogenic differentiation of rabbit mesenchymal stromal cells in vitro. We evaluated its efficacy as a regenerative interpositional material applied after bony bar resection in a rabbit model of growth plate injury. Radiographic imaging was used to monitor limb length and tibial plateau angle, microcomputed tomography assessed bone morphology, and histology characterized the repair tissue that formed. Our 3D printed growth plate mimetic composite resulted in improved tibial lengthening compared to an untreated control, cartilage-mimetic hydrogel only condition, and a fat graft. However, in vivo the 3D printed growth plate mimetic composite did not show cartilage regeneration within the construct histologically. Nevertheless, this study demonstrates the feasibility of a 3D printed biomimetic composite to improve limb lengthening, a key functional outcome, supporting its further investigation as a treatment for growth plate injuries.