with high throughput. [21][22][23] By interrupting the fluid flow during the exposure through an interference phase mask, the stop-flow lithography technique enables high-resolution fabrication of patterned particles. [24][25][26] In combination with digital mirror devices, the vertical-flow lithography (VFL) method can synthesize anisotropic particles in real time using a light source that is aligned with the fluid flow. [27,28] To overcome the limiting resolution of these projection techniques, scanning two-photon lithography is coupled with CFL for the synthesis of truly 3D particles and fibers. [29,30] The current two-photon CFL (TP-CFL) methods offer fast in-plane laser writing speeds surpassing 1 mm s −1 using scanning galvanometer mirrors; however, the out-of-plane writing speed is still limited by the piezo-driven nanostage movement of typically 100 µm s −1 . [31] The transfer of the continuous real-time synthesis from particles (0D), fibers (1D), and planar geometries (2D) toward microtubes (3D) represents an intricate challenge with respect to the synthesis of i) high aspect ratio microtubes with ii) rigorous control over the morphology and surface topology by coupling scanning two-photon lithography with a vertical flow to obtain iii) high in-plane and out-of-plane synthesis speeds with iv) sub-micrometer resolution. Below, for the first time, we demonstrate two-photon VFL (TP-VFL) that combines the advantages of TP-CFL with VFL to control the out-of-plane fabrication speed by the vertical flow inside a microfluidic channel. This unique approach is an enabling step toward the synthesis of tubular scaffolds for vascular tissue engineering, microstents for intravascular pressure reduction, microneedles for transdermal drug delivery, nerve guides for neuronal regeneration, and porous hollow fiber membranes for separation applications. [32][33][34][35][36] Results and Discussion Fluid Flow-Coupled Two-Photon PolymerizationTo validate the proposed hypothesis, we design and fabricate a microfluidic chip featuring precise fluid-flow control (Figure 1). The designed master mold (Figure 1a) is printed onto a glass slide using maskless dip-in laser lithography (Figure 1b). [37,38] Soft-lithography replica molding is used to obtain the silicone microfluidic chip plasma bonded to a glass slide (Figure 1c). [39] Two-photon vertical-flow lithography is demonstrated for synthesis of complex-shaped polymeric microtubes with a high aspect ratio (>100:1). This unique microfluidic approach provides rigorous control over the morphology and surface topology to generate thin-walled (<1 µm) microtubes with a tunable diameter (1-400 µm) and pore size (1-20 µm). The interplay between fluid-flow control and two-photon lithography presents a generic high-resolution method that will substantially contribute toward the future development of biocompatible scaffolds, stents, needles, nerve guides, membranes, and beyond. Lithography

We describe an impedance-based method for cell barrier integrity testing. A four-electrode electrical impedance spectroscopy (EIS) setup can be realized by simply connecting a commercial chopstick-like electrode (STX-1) to a potentiostat allowing monitoring cell barriers cultivated in transwell inserts. Subsequent electric circuit modeling of the electrical impedance results the capacitive properties of the barrier next to the well-known transepithelial electrical resistance (TEER). The versatility of the new method was analyzed by the EIS analysis of a Caco-2 monolayer in response to (a) different membrane coating materials, (b) two different permeability enhancers ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and saponin, and (c) sonoporation. For the different membrane coating materials, the TEERs of the standard and new protocol coincide and increase during cultivation, while the capacitance shows a distinct maximum for three different surface materials (no coating, Matrigel ® , and collagen I). The permeability enhancers cause a decline in the TEER value, but only saponin alters the capacitance of the cell layer by two orders of magnitude. Hence, cell layer capacitance and TEER represent two independent properties characterizing the monolayer. The use of commercial chopstick-like electrodes to access the impedance of a barrier cultivated in transwell inserts enables remarkable insight into the behavior of the cellular barrier with no extra work for the researcher. This simple method could evolve into a standard protocol used in cell barrier research.

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