Fig. 12

Effect of defocus step. Experimentally measured potential well depths depend on the applied defocus step (the reconstruction is performed using three images only). Although, the difference of the potential variation due to defocus variation is not the same for different microscopes, a clear trend is visible in the graph. The measured potential well depth decreases with increasing defocus step in a non-linear dependency. As the remaining misalignment of the focal series becomes more severe with increasing defocus steps, the resulting potential variation is damped more strongly leading to an underestimation of the real potential well if large defocus steps are applied. In contrast, if small defocus steps are used, the accuracy is limited by the effects of noise. Therefore, a direct comparison of results obtained on different microscopes or based on different defocus steps is not possible