Confocal imaging characterization of two-photon lithography microstructures for cell cultures

Two-photon lithography (2PL) is a direct laser writing approach for micro- and nanofabrication of 3D polymeric structures for a variety of applications. Due to its high resolution (<100nm), inspection of actual dimensions of realized features is best performed via scanning electron microscopy (SEM). However, SEM analysis is not always feasible, as in the case of structural deformations caused by solvent drying, or in applications in which the structures should stay in liquid environments during the whole course of the experiment. This latter is the case of 3D polymeric elements for cell mechanical studies or microfluidic applications, requiring stable architectures for long periods in liquid even when strong deformations are applied. For these reasons, SEM analysis needs to be replaced with other techniques able to obtain high-resolution measurements and analysis of in-liquid micrometric objects. Here we evaluate confocal microscopy to achieve quantitative volumetric imaging of 3D polymeric structures in liquid. With sample measurements of both axial and lateral voxel dimensions in 3D cage-like structures used for studying the invasiveness of cell cultures [Sci. Rep. 5, 10531, 2015], we conclude that this approach can be considered a valid alternative to SEM for experiments involving evaluation of structural integrity in liquid environments.