Single-molecule localization software applied to photon counting imaging.

Centroiding in photon counting imaging has traditionally been accomplished by a single-step, noniterative algorithm, often implemented in hardware. Single-molecule localization techniques in superresolution fluorescence microscopy are conceptually similar, but use more sophisticated iterative software-based fitting algorithms to localize the fluorophore. Here, we discuss common features and differences between single-molecule localization and photon counting imaging and investigate the suitability of single-molecule localization software for photon event localization. We find that single-molecule localization software packages designed for superresolution microscopy-QuickPALM, rapidSTORM, and ThunderSTORM-can work well when applied to photon counting imaging with a microchannel-plate-based intensified camera system: photon event recognition can be excellent, fixed pattern noise can be low, and the microchannel plate pores can easily be resolved.