Light-sheet confined super-resolution using two-photon photoactivation

Light-sheet microscopy is a useful tool for performing biological investigations of thick samples and it has recently been demonstrated that it can also act as a suitable architecture for super-resolution imaging of thick biological samples by means of individual molecule localization. However, imaging in depth is still limited since it suffers from a reduction in image quality caused by scattering effects. This paper sets out to investigate the advantages of non-linear photoactivation implemented in a selective plane illumination configuration when imaging scattering samples. In particular, two-photon excitation is proven to improve imaging capabilities in terms of imaging depth and is expected to reduce light-sample interactions and sample photo-damage. Here, two-photon photoactivation is coupled to individual molecule localization methods based on light-sheet illumination (IML-SPIM), allowing super-resolution imaging of nuclear pH2AX in NB4 cells.