Image scanning microscopy with a long depth of focus generated by an annular radially polarized beam.

Image scanning microscopy (ISM) is a promising tool for bioimaging owing to its integration of signal to noise ratio (SNR) and super resolution superior to that obtained in confocal scanning microscopy. In this paper, we introduce the annular radially polarized beam to the ISM, which yields an axially extended excitation focus and enhanced resolution, providing a new possibility to obtain the whole information of thick specimen with a single scan. We present the basic principle and a rigorous theoretical model for ISM with annular radially polarized beam (ISM-aRP). Results show that the resolution of ISM-aRP can be enhanced by 4% compared with that in conventional ISM, and the axial extent of the focus is longer than 6λ. The projected view of the simulated fluorescent beads suspension specimen demonstrates the validity of ISM-aRP to obtain the whole information of volume sample. Moreover, this simple method can be easily integrated into the commercial laser scanning microscopy systems.