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 twofold, it has since been extended to enhance axial resolution twofold (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 nonlinear, 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.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.
Highlights
Structured illumination microscopy (SIM) is a method in fluorescence microscopy which is capable of providing resolutions better than those that can be achieved using uniform illumination in epifluorescence wide-field microscopy
We argue that image scanning microscopy (ISM), while experimentally a different method, is a form of SIM and show how arbitrary illumination patterns can be used (§9)
These objectives can form a pattern right at the coverslip boundary at the limit of their numerical apertures and have the near-field be patterned with the same spatial period, even though there is no way of creating such a fine pattern in water using far-field illumination
Summary
Cite this article: Manton JD. 2022 Answering some questions about structured illumination microscopy. Demonstrated in 1999 to enhance the lateral resolution twofold, it has since been extended to enhance axial resolution twofold (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.
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