3D printed vascular phantoms for high-resolution biophotonic image quality assessment via direct laser writing.

Fluorescence imaging techniques such as fluorescein angiography and fundus autofluorescence are often used to diagnose retinal pathologies; however, there are currently no standardized test methods for evaluating device performance. Here we present microstructured fluorescent phantoms fabricated using a submicron-scale three-dimensional printing technology, direct laser writing (DLW). We employ an in situ DLW technique to print 10 µm diameter microfluidic channels that support perfusions of fluorescent dyes. We then demonstrate how broadband photoresist fluorescence can be exploited to generate resolution targets and biomimetic models of retinal vasculature using standard DLW processes. The results indicate that these approaches show significant promise for generating better performance evaluation tools for fluorescence microscopy and imaging devices.

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies.

Colorimetrical uncertainty estimation for the performance assessment of whole slide imaging scanners.

Purpose: Whole slide imaging (WSI) scanners produce tissue slide images with a large field of view and a high resolution for pathologists to use in diagnoses. Color performance tests of these color imaging devices are necessary and can use stained tissue slides if the color truth is established using a hyperspectral imaging microscopy system (HIMS). The purpose of this study was to estimate the reproducibility uncertainty of CIELAB coordinates for a reference tissue slide measured by both the HIMS and a WSI scanner. Approach: We compared the color performances of the WSI scanner to those of the reference established by the HIMS using the International Commission on Illumination (Commission Internationale de l'Éclairage, or CIE) 1976 Δ E a b * color difference with the just noticeable color difference (JNCD, Δ E a b * ≤ 2 ), and the results from the overlap of the CIELAB coordinates' uncertainty within the error bar, with a coverage factor k = 2 . The reported uncertainty results from measurements and image registration uncertainties. Results: For the blank area common to the HIMS and the WSI average images, the color agreement was higher using the JNCD condition versus the CIELAB uncertainty overlap criterion (82% and 20% of the pixels in the images, respectively). This difference is explained by the fact that numerous pixels have CIELAB coordinates near one another but corresponding to CIELAB uncertainty values small enough not to overlap. In the colored area of the images, the JNCD condition was met for 0.19% of the pixels in the images, compared with 4.3% for the CIELAB uncertainty overlap criterion. Conclusions: The distribution of uncertainties on the CIELAB coordinates was broader for the HIMS compared with the WSI scanner. The WSI scanner had a systemic error in the color reproduction, which pointed to a potential inadequate color calibration of this device.

Geometric Determinants of In-Situ Direct Laser Writing.

Direct laser writing (DLW) is a three-dimensional (3D) manufacturing technology that offers significant geometric versatility at submicron length scales. Although these characteristics hold promise for fields including organ modeling and microfluidic processing, difficulties associated with facilitating the macro-to-micro interfaces required for fluid delivery have limited the utility of DLW for such applications. To overcome this issue, here we report an in-situ DLW (isDLW) strategy for creating 3D nanostructured features directly inside of-and notably, fully sealed to-sol-gel-coated elastomeric microchannels. In particular, we investigate the role of microchannel geometry (e.g., cross-sectional shape and size) in the sealing performance of isDLW-printed structures. Experiments revealed that increasing the outward tapering of microchannel sidewalls improved fluidic sealing integrity for channel heights ranging from 10 µm to 100 µm, which suggests that conventional microchannel fabrication approaches are poorly suited for isDLW. As a demonstrative example, we employed isDLW to 3D print a microfluidic helical coil spring diode and observed improved flow rectification performance at higher pressures-an indication of effective structure-to-channel sealing. We envision that the ability to readily integrate 3D nanostructured fluidic motifs with the entire luminal surface of elastomeric channels will open new avenues for emerging applications in areas such as soft microrobotics and biofluidic microsystems.

A facile multi-material direct laser writing strategy.

Direct laser writing (DLW) is a three-dimensional (3D) manufacturing technology that offers vast architectural control at submicron scales, yet remains limited in cases that demand microstructures comprising more than one material. Here we present an accessible microfluidic multi-material DLW (µFMM-DLW) strategy that enables 3D nanostructured components to be printed with average material registration accuracies of 100 ± 70 nm (ΔX) and 190 ± 170 nm (ΔY) - a significant improvement versus conventional multi-material DLW methods. Results for printing 3D microstructures with up to five materials suggest that µFMM-DLW can be utilized in applications that demand geometrically complex, multi-material microsystems, such as for photonics, meta-materials, and 3D cell biology.

Layer-by-layer assembly of pH-responsive, compositionally controlled (co)polyelectrolytes synthesized via RAFT.

Homo- and block copolyelectrolytes that have well-defined structures and are responsive to pH were synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization and employed to produce layer-by-layer (LBL) films. Acrylamido monomers with carboxylate, sulfonate, and amine functionality were utilized to provide both strong and weak homopolyelectrolytes and mixed strong/weak copolyelectrolyte systems. Multilayer films were prepared under specified conditions of pH and ionic strength and analyzed via atomic force microscopy and ellipsometry to study the effects of changes in the local molecular environment on film morphologies. The pH responsiveness and integrity of the multilayer assemblies were investigated by exposing films to solutions of varying pH in a fluid cell and performing in situ AFM analysis. The multilayer dimensions, morphology, and integrity were found to depend on the molecular architecture of the polyelectrolytes, with changes in segmental type and repeating unit distribution producing dramatic differences in film characteristics. These results suggest the possibility of producing LBL assemblies of precisely controlled dimensions and properties by specifically tailoring copolymer structure. To our knowledge, this is the first report of LBL assembly of RAFT-synthesized homo- and copolyelectrolyte multilayer complexes.