Answering some questions about structured illumination microscopy

TL;DR

This paper clarifies the principles, limitations, and advancements of structured illumination microscopy (SIM), emphasizing the need for non-polynomial responses for true super-resolution and offering a new reconstruction method.

Contribution

It provides a comprehensive overview of SIM, addresses common misconceptions, introduces a real-space reconstruction approach, and analyzes its development over two decades.

Findings

SIM is still fundamentally diffraction-limited under normal use.

A non-polynomial response is necessary for true super-resolution in SIM.

A new real-space reconstruction method is proposed for SIM and related techniques.

Abstract

Structured illumination microscopy (SIM) provides images of fluorescent objects at an enhanced resolution greater than that of conventional epifluorescence wide-field microscopy. Initially demonstrated in 1999 to enhance the lateral resolution two-fold, it has since been extended to enhance axial resolution two-fold (2008), applied to live-cell imaging (2009) and combined with myriad other techniques, including interferometric detection (2008), confocal microscopy (2010) and light sheet illumination (2012). Despite these impressive developments, SIM remains, perhaps, the most poorly understood `super-resolution' method. In this article, we provide answers to the 13 questions regarding SIM proposed by Prakash et al., along with answers to a further three questions. After providing a general overview of the technique and its developments, we explain why SIM as normally used is still diffraction-limited. We then highlight the necessity for a non-polynomial, and not just non-linear, response to the illuminating light in order to make SIM a true, diffraction-unlimited, super-resolution technique. In addition, we present a derivation of a real-space SIM reconstruction approach that can be used to process conventional SIM and image scanning microscopy (ISM) data and extended to process data with quasi-arbitrary illumination patterns. Finally, we provide a simple bibliometric analysis of SIM development over the past two decades and provide a short outlook on potential future work.