The voxel onset time as an in situ method to evaluate focal position effects on two-photon-induced lithography

Two-photon-induced lithography is a versatile method to generate arbitrary three-dimensional microstructures. Although the lithographic result sensitively depends on the experimental conditions, there is a lack of in situ methods to measure process conditions prior to structuring. Current methods rely on determining the size of cross-linked structures, such as single-volume pixels (voxels), as a result of a set of parameters. This procedure is time consuming and possesses several inherent drawbacks, since results are not easily interpretable. Therefore, we established an in situ method, called the voxel onset time (VOT) method, which is easy to integrate in an existing two-photon lithographic setup and is based on determining the time that a voxel necessitates to form by measuring the transmitted laser intensity. In this study, we demonstrate how the VOT method can be used to determine the influence of the axial focal position on voxel formation for different experimental conditions. We find that the voxel onset time is strongly linked to the maximum intensity that is influenced by specimen-induced spherical aberration, especially for a high-numerical-aperture objective.