Post-processing strategies in image scanning microscopy.

Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view.