@inbook{7941,
  abstract     = {Expansion microscopy is a recently developed super-resolution imaging technique, which provides an alternative to optics-based methods such as deterministic approaches (e.g. STED) or stochastic approaches (e.g. PALM/STORM). The idea behind expansion microscopy is to embed the biological sample in a swellable gel, and then to expand it isotropically, thereby increasing the distance between the fluorophores. This approach breaks the diffraction barrier by simply separating the emission point-spread-functions of the fluorophores. The resolution attainable in expansion microscopy is thus directly dependent on the separation that can be achieved, i.e. on the expansion factor. The original implementation of the technique achieved an expansion factor of fourfold, for a resolution of 70–80 nm. The subsequently developed X10 method achieves an expansion factor of 10-fold, for a resolution of 25–30 nm. This technique can be implemented with minimal technical requirements on any standard fluorescence microscope, and is more easily applied for multi-color imaging than either deterministic or stochastic super-resolution approaches. This renders X10 expansion microscopy a highly promising tool for new biological discoveries, as discussed here, and as demonstrated by several recent applications.},
  author       = {Truckenbrodt, Sven M and Rizzoli, Silvio O.},
  booktitle    = {Methods in Cell Biology},
  isbn         = {978012820807-6},
  issn         = {0091-679X},
  pages        = {33--56},
  publisher    = {Elsevier},
  title        = {{Simple multi-color super-resolution by X10 microscopy}},
  doi          = {10.1016/bs.mcb.2020.04.016},
  volume       = {161},
  year         = {2021},
}

@article{6052,
  abstract     = {Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60–80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.},
  author       = {Truckenbrodt, Sven M and Sommer, Christoph M and Rizzoli, Silvio O and Danzl, Johann G},
  journal      = {Nature Protocols},
  number       = {3},
  pages        = {832–863},
  publisher    = {Nature Publishing Group},
  title        = {{A practical guide to optimization in X10 expansion microscopy}},
  doi          = {10.1038/s41596-018-0117-3},
  volume       = {14},
  year         = {2019},
}

@article{145,
  abstract     = {Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non-recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.},
  author       = {Truckenbrodt, Sven M and Viplav, Abhiyan and Jähne, Sebsatian and Vogts, Angela and Denker, Annette and Wildhagen, Hanna and Fornasiero, Eugenio and Rizzoli, Silvio},
  issn         = {0261-4189},
  journal      = {The EMBO Journal},
  number       = {15},
  publisher    = {Wiley},
  title        = {{Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission}},
  doi          = {10.15252/embj.201798044},
  volume       = {37},
  year         = {2018},
}

@article{6499,
  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.},
  author       = {Truckenbrodt, Sven M and Maidorn, Manuel and Crzan, Dagmar and Wildhagen, Hanna and Kabatas, Selda and Rizzoli, Silvio O},
  issn         = {1469-3178},
  journal      = {EMBO reports},
  number       = {9},
  publisher    = {EMBO},
  title        = {{X10 expansion microscopy enables 25‐nm resolution on conventional microscopes}},
  doi          = {10.15252/embr.201845836},
  volume       = {19},
  year         = {2018},
}