news & views discovery is sure to inspire further research efforts in this direction. Once the synthetic strategy has been refined into a more general method, it will undoubtedly shift the existing retrosynthetic paradigm towards complex nitrogen-containing heterocycles.  ❐ Olga V. Zatolochnaya and Vladimir Gevorgyan are in the Department of Chemistry at University of

Illinois at Chicago, Chicago, Illinois, 60607, USA. e-mail: [email protected] References

1. Jeffrey, J. L. & Sarpong, R. Chem. Sci. 4, 4092–4106 (2013). 2. Espino, C. G. & Du Bois, J. Angew. Chem. Int. Ed. 40, 598–600 (2001). 3. Roizen, J. L., Harvey, M. E. & Du Bois, J. Acc. Chem. Res. 45, 911–322 (2012). 4. Rice, G. T. & White, M. C. J. Am. Chem. Soc. 131, 11707–11711 (2009).

5. Nadres, E. T. & Daugulis, O. J. Am. Chem. Soc. 134, 7–10 (2012). 6. He, G., Zhao, Y., Zhang, S., Lu, C. & Chen, G. J. Am. Chem. Soc. 134, 3–6 (2012). 7. Neumann, J. J., Rakshit, S., Dröge, T. & Glorius, F. Angew. Chem., Int. Ed. 48, 6892–6895 (2009). 8. Wang, Z., Ni, J., Kuninobu, Y. & Kanai, M. Angew. Chem. Int. Ed. 53, 3496–3499 (2014). 9. Nguyen, Q., Sun, K. & Driver, T. G. J. Am. Chem. Soc. 134, 7262–7265 (2012). 10. Hennessy, E. T. & Betley, T. A. Science 340, 591–595 (2013). 11. McNally, A., Haffemayer, B., Collins, B. S. L. & Gaunt, M. J. Nature 510, 129–133 (2014).

FLUORESCENCE MICROSCOPY

Strategic blinking

For decades chemists have focused on increasing the brightness of fluorophores. In super-resolution microscopy, however, fluorophores that preferentially exist in a non-fluorescent state, but occasionally re-arrange into a fluorescent form, can give better results.

Gražvydas Lukinavičius and Kai Johnsson

S

uper-resolution microscopy — the imaging of objects with a resolution below the diffraction barrier of light — has enabled numerous important discoveries in biology 1. One approach is single-molecule localization microscopy (SLM), in which individual fluorophores are separated by stochastically switching them between fluorescent ‘on’ and non-fluorescent ‘off ’ states. At any given time, only a sparse subset of fluorophores are in the on state, and therefore detected and localized. Repeating this process thousands of times enables reconstruction of an image with a resolution below the diffraction barrier 1. One of the challenges of SLM is to force fluorophores, which have been developed to be as bright and photostable as possible2, into an ‘of ’ state. This is generally achieved by illuminating samples with high-intensity light; however, the resulting phototoxicity can be problematic for live-cell microscopy 3. Another problem is that organic dyes that are well suited for SLM such as Cy5 or Alexa6471,4 are not membrane permeable and thus not well suited for live-cell imaging. Furthermore, efficient switching of these dyes requires the presence of oxygen-scavenging systems and/or thiols4. In this issue of Nature Chemistry, Yasuteru Urano, Mako Kamiya and coworkers report a new class of fluorophores that exist in a non-fluorescent off state for more than 99% of the time, and only occasionally and for very short times (hundreds of milliseconds) spontaneously rearrange into a highly fluorescent form5. The probes are excited in the far-red light, are membrane permeable and do not require any additives for efficient switching.

a

b O N

Si

N

Off Spontaneous blinking

OH

N

Si

N

On

Figure 1 | Construction of super-resolution images using the hydroxymethyl silicon-rhodamine (HMSiR) dye. a, HMSiR shown in its fluorescent ‘on’ (red) and predominant non-fluorescent ‘off’ (black) state. b, A super-resolution image of microtubules labelled with HMSiR. Scale bar, 2 μm.

Together, these properties make the probes ideally suited for live-cell SLM. To achieve this, Urano and colleagues developed appropriately substituted rhodamine derivatives that can spontaneously rearrange into a non-fluorescent spirocyclic form. By carefully adjusting the reactivity of the nucleophile and electrophile responsible for spirocyclization they discovered HMSiR (Fig. 1a), a hydroxymethyl silicon-rhodamine derivative that under physiological conditions predominantly exists as a spirocyclic compound and only occasionally reveals its brighter side. The

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potential of such a molecule for live-cell super-resolution microscopy is obvious. However, if the probe cannot be specifically attached to selected proteins inside living cells then its utility would be greatly limited. To clear this final hurdle for live-cell applications, the team made use of selflabelling protein tags such as SNAP-tag and Halo-tag, which permit the specific coupling of synthetic dyes to fusion proteins in living cells. This approach enabled HMSiR to be converted into a SNAP and Halo-tag substrate and to be subsequently used for the specific labelling of fusion proteins in living cells. 663

news & views With these excellent results in hand, Urano and colleagues used the SNAP-tag and Halo-tag substrates of HMSiR to label microtubules (Fig. 1b). This subsequently enabled the dynamics of polymerization and depolymerization of microtubules in living cells to be imaged with a spatial resolution of about 50 nm and at a temporal resolution of 15 s per image. Although such spatiotemporal resolution in live-cell imaging of microtubules has been achieved previously by others1, those studies required much higher light intensities than used here: only 40 W cm–2 illumination power of red light was applied by Urano and colleagues whereas in conventional SLM approaches light intensities of around 1,000 W cm–2 are used. Such dim light permitted the researchers to perform live-cell imaging over periods of 60 minutes without any obvious phototoxicity. Another attractive feature of these probes for live-cell imaging is that their spontaneous blinking does not require the addition of any additives into

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the medium. The spontaneous blinking of HMSiR furthermore enables the acquisition of super-resolution images with standard fluorescence microscopes found in most biological labs. We can expect that HMSiR is only the first of a new generation of SLM probes based on spirocyclization. Urano and colleagues have already indicated that it should be feasible to generate probes with different colours, which would be of great interest for bioimaging. Furthermore, this work reminds us how precisely controlled spirocyclization of xanthene derivatives can be exploited to generate a variety of sophisticated fluorescent probes. For example, probes for image-guided surgery have been generated in which the spirocyclic state is reversed through modification by gamma-glutamyltranspeptidase, a prominent tumour marker 6. Using similar design principles, probes have been generated in which binding to their intracellular targets suppresses

spirocyclization, resulting in the generation of powerful fluorogenic probes7. This degree of sophistication in probe design is an important prerequisite to help bioimaging to move from the imaging of fixed and static samples, to the super-resolution imaging of dynamic components in living systems. Chemists need to lend biologists a hand to achieve this important goal. ❐ Gražvydas Lukinavičius and Kai Johnsson are in the École Polytechnique Fédérale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering, NCCR Chemical Biology, 1015 Lausanne, Switzerland. e-mail: [email protected] References 1. Klein, T., Proppert, S. & Sauer, M. Histochem. Cell Biol. 6, 561–575 (2014). 2. Lavis, L. D. & Raines, R. T. ACS Chem. Biol. 9, 855–866 (2014). 3. Schneckenburger, H. et al. J. Microsc. 245, 311–318 (2012). 4. Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. & Zhuang, X. Nature Methods 8, 1027–1036 (2011). 5. Uno, S.-n. et al. Nature Chem. 6, 681–689 (2014). 6. Urano, Y. et al. Sci. Transl. Med. 3, 110–119 (2011). 7. LukinaviČius, G. et al. Nature Methods 11, 731–733 (2014).

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Fluorescence microscopy: strategic blinking.

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