Answers to fundamental questions in superresolution microscopy

Abstract

This article presents answers to the questions on superresolution and structured illumination microscopy (SIM) as raised in the editorial of this collection of articles ( https://doi.org/10.1098/rsta.2020.0143 ). These answers are based on my personal views on superresolution in light microscopy, supported by reasoning. Discussed are the definition of superresolution, Abbe's resolution limit and the classification of superresolution methods into nonlinear-, prior knowledge- and near-field-based superresolution. A further focus is put on the capabilities and technical aspects of present and future SIM methods. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

Answering some questions about structured illumination microscopy

“…In this article, we aim to provide a general overview of SIM and answer the 13 questions recently proposed by Prakash et al [ 19 ] (§§1–13), along with three more of our own invention (§§14–16). A similar attempt at answering the 13 questions of Prakash et al has recently been presented by Heintzmann [ 20 ]. For some questions, the answers given by Heintzmann are significantly different from those presented here: where this is the case, this will be highlighted in later sections.…”

“… Super resolution —the subset of enhanced resolution methods not limited by diffraction and theoretically capable of infinite resolution, discussed further in §4, such as stimulated emission depletion microscopy and saturated SIM. Note that some authors, including Heintzmann in his answers to these questions [ 20 ], would classify all methods capable of imaging beyond the extended resolution limit as super-resolution methods, but this is not a sufficient condition for ‘breaking’ the diffraction barrier [ 37 ]. Furthermore, it is worth highlighting the difference between ‘imaging at the diffraction limit’, i.e.…”

“…It is particularly instructive to compare this answer (yes) with the companion given by Heintzmann: ‘a clear “no”’ [ 20 ]. For Heintzmann, taking equation ( 2.17 ), all forms of SIM break the emission Abbe limit and hence break the diffraction limit.…”

Super-resolution structured illumination microscopy: past, present and future

“…The following questions have not always been agreed upon in the super-resolution/SIM field, and with this special issue, we hope to address some of these: For answers to these fundamental questions in super-resolution microscopy, please refer to [85].…”

At the molecular resolution with MINFLUX?

“…The major advantage of the direct combination is in resolution enhancement by increasing the information per photon. For readers interested in the technical details of these methods, following are some excellent recent reviews on this topic [15][16][17][18][19]. MINFLUX and SIMFLUX have demonstrated a resolution of less than 10 nm on synthetic structures like DNA origami and over 40 nm on biological structures.…”