Image scanning microscopy (ISM) aims to improve on confocal scanning microscopy by replacing the traditional detection pinhole with a detector array. A confocal pinhole offers improved image resolution and depth sectioning, but at the cost of discarding the photons that fall outside this small aperture. In ISM, the pinhole is replaced with an imaging array, allowing the central pixel(s) to act in a manner similar to a traditional pinhole, while the outer pixels capture light that would otherwise be lost. In one of the simpler ISM configurations, the light in these outer pixels can be used to characterize contributions from out-of-focus depths, and by subsequent subtraction of these contributions, depth sectioning and contrast may be improved.

More generally, ISM provides a rich multi-dimensional dataset for each scan location. This allows flexibility to consider more exotic laser illumination patterns, with image reconstruction approached systematically using frameworks such as maximum likelihood estimation. In this paper, Sheppard et. al detail a framework for evaluation of ISM performance, by building on their previous analysis of doughnut-beam illumination ISM. Clear metrics are applied for the overall signal level, background contributions, and optical sectioning; and by deriving tractable optical models for confocal and ISM microscopes (with Airy-disk or doughnut-beam illumination) the relative benefits of each can be quantitatively compared. This article gives a clear characterization of the advantages of doughnut-illuminated ISM, and will be useful to anyone looking to understand the performance of the modality.

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