Coda: Trade-Offs, Cautions, and Limitations of Superresolution Optical Microscopes
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My aim in this chapter is to encourage critical skeptical thinking about superresolution microscopy. It is important to note that the precision and accuracy of single-molecule localization are different concepts. Typically, authors report the precision of localization of a single molecule. That is not equivalent to the accuracy of localization. The accuracy of localization is a measure of the fidelity with which single-molecule localization is identical to the actual location within the specimen. To assess the accuracy of superresolution single-molecule locations and therefore the final superresolution image we require calibration specimens whose structures are independently determined. An alternative is to use correlative microscopy (e.g., transmission electron microscopy) to validate the accuracy of information obtained from the superresolution image. Localization techniques achieve high precision in localization of a fluorescent molecule. The size of a fluorescent molecule affects the accuracy of the position of the structure to which the label is attached. This is of increasing importance as the size of the label, including its associated linker molecules, approaches the required resolution of the superresolution image.
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