Abstract
Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.
Highlights
The resolution of fluorescence microscopes has been limited by the diffraction barrier to approximately half of the wavelength of the imaging light
This barrier has been lifted by several microscopy concepts, for example, by using patterned light beams to determine the coordinates from which fluorophores are permitted to emit, as in the stimulated emission depletion (STED) family [1,2], or by determining the positions of single fluorophores that emit randomly, as in photo-activated localization microscopy (PALM) [3], stochastic optical reconstruction microscopy (STORM and dSTORM) [4,5], or ground state depletion microscopy followed by individual molecule return (GSDIM) [6] such technologies have been applied to biology for more than a decade, their general impact on biomedical research is still relatively limited
The initial implementations of this approach were performed with gels that expanded, on average, about fourfold, which resulted in lateral resolutions of ~70 nm, i.e. not as high as that of modern STED or STORM microscopes [7]
Summary
The resolution of fluorescence microscopes has been limited by the diffraction barrier to approximately half of the wavelength of the imaging light (in practice, 200–350 nm). This type of analysis can be performed in confocal microscopy, but one does not necessarily obtain much information (Fig EV4), as the dim samples obtained by 1,000-fold volume expansion are not ideal for confocal imaging.
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