Two photon lithography allows the fabrication of arbitrary 3D structures with possible applications as micromechanical and microelectromechanical systems, photonic devices, 3D cell culture systems and scaffolds for tissue engineering. In order to achieve maximum resolution the process parameters have to be perfectly fitted to a given material. Normally, this interaction is studied by measuring the size of the generated volume pixels (voxel). In general, these analyses are time consuming, since

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... hey necessitate careful sample preparation and the use of a scanning electron microscope. In this paper, the threshold time for voxel formation, the voxel onset time (VOT), is presented as a parameter, which can give additional insight in the process of voxel formation. VOT is measured by a simple optical method, which can be implemented easily in already existing two photon lithography setups. Since the VOT method is considerably faster than voxel size analysis, it could be used in the future for faster screening of novel materials, while giving additional input of time dependencies of voxel growth. Introducion Two photon lithography (TPL), or direct laser writing is a versatile tool to generate arbitrary 3D structures with subdiffraction limited resolution [1, 2, 3, 4] . A tightly focused laser beam delivers high photon densities in a confined space and time. The resulting high photon densities trigger a nonlinear optical effect in a photosensitive material, resulting in confined chemical polymerization, crosslinking, or photoactivation. Applications range from photonics to biomedicine [5, 6, 7, 8, 9, 10, 11, 12] . Two main challenges are encountered at opposite sides of the size scale. On the one side, smaller feature sizes are desired, in order to enhance the functionality of the generated object. In the last couple of years, different strategies, such as stimulated emission depletion (STED) lithography, have been pursuit to achieve sub-100 nm feature resolution [13, 14, 15, 16] . On the other hand, macroscopic products with high resolution features are necessary to translate this promising fabrication technology from the laboratory into industry. Enhancing the sensitivity of the employed material and parallelization through multifocal technologies are the two main strategies to achieve this aim [17, 18, 19, 20] . In order to address these two challenges, achievable resolution and fabrication speed, a deep understanding of the interaction of laser irradiation and photosensitive material system is necessary. This interaction has been described mostly by measuring the size of crosslinked structures under different process conditions [21, 22, 23, 24] . Single volume pixels (voxels) represent a snapshot of the polymerization process at a given time. Thus, their length and diameter have been used to study polymerization efficiency, kinetics and overall de-pendency on process parameters. However, this type of voxel analysis does not directly allow studying the time dependency of single voxel formation and for a kinetic study massive amounts of voxels have to be prepared and analyzed. Yet, voxel growth kinetic has a profound effect on minimal achievable resolution, as well as on possible process speed. In this study, a method is presented, which measures voxel formation in-situ. In the case of radical induced polymerization, voxel formation is a process based on local crosslinking of a prepolymer, initiated by a photosensitive molecule called photoinitiator. This crosslinking results in a local change in density and therefore refractive index. With ongoing voxel formation the size of the crosslinked volume increases, as well as the crosslinking density. Structures possessing a different refractive index than their surrounding lead to light scattering and refraction. Scattering strongly depends on particle size and refractive index and is therefore a feasible tool to characterize voxel growth in situ. In this study, the threshold time for voxel formation, the voxel onset time (VOT), is measured based on the above described strategy. Materials and Methods The experimental setup for TPL ( Fig. 1) consists of a tunable Ti:Sapphire laser source, with a pulse duration of approximately 100 fs, a repetition rate of 80 MHz and a maximum mean laser power output of approximately 3 W. The laser beam passes a λ/2 waveplate and a polarizing beam splitter for attenuation, a mechanical shutter and a telescope, before being coupled in a microscope objective (NA