RESEARCH HIGHLIGHTS

IMAGING Death by super-resolution imaging

Researchers report that the illumination Sauer notes that just the 26-nm difference intensities used in super-resolution imag- between 488 nm and 514 nm can be the ing can irreversibly damage live cells. difference between life and death for cells Super-resolution imaging methods such being imaged. as direct stochastic optical reconstruction Sauer wants a take-home message from (dSTORM), photoactivated this work to be that “users should carefully localization microscopy (PALM) and stimu- dose the use of wavelengths shorter than 500 lated emission depletion (STED) microscopy nm for photoactivation or photoconversion, can provide highly detailed views of struc- even at very low intensities. The longer the tures within cells. However, these methods irradiation wavelength, the better the sur- require high doses of light, which can have vival chances of cells.” This may be particu- unwanted effects, known as photodamage larly relevant to PALM experiments, which and phototoxicity, on biological samples. use near-UV light to photoactivate fluoro- Markus Sauer at the University of Würzburg, phores over multiple rounds of imaging. along with two graduate students in his labo- The authors made other important ratory, Sina Wäldchen and Julian Lehmann, Image of a `frozen’ mammalian cell where the observations in the course of their experi- sought to carefully characterize such effects top portion was treated by light, destroying the ments. For example, they found that after in live cells. network. Reprinted from Wäldchen et al. irradiation, some cells underwent apopto- The research was motivated by con- (2015). sis before death, whereas others bypassed flicting reports regarding phototoxicity in dead and living cells. Some of their results that step. These latter, so-called frozen cells the field of super-resolution were unexpected. In particular, Sauer says were immediately killed and fixed in place microscopy. “Some people claimed that he was surprised that “dyes contribute to by the light treatment. Further analysis

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature very low light doses, even sunlight, can phototoxicity, but it is mainly the direct showed that, despite looking unchanged, seriously kill cells,” says Sauer, whereas interaction of the light with other intrinsic the membrane and cytoskeleton of frozen “other researchers use orders-of-magnitude cellular molecules which is responsible for cells were destroyed by the light treatment. higher light doses in living cells and even in damage.” They found that transfection can These results suggest that examination for npg living mouse brain and claim that they do increase cells’ photosensitivity relative to phototoxic effects should not be limited to not observe any signs of photodamage.” The that of untreated controls, as can the intro- phenotypic assessment immediately after team was interested in the effects of fluoro- duction of a fluorophore. They also discov- imaging. phores, which are known to generate harm- ered that cell lines differ in their photosen- These results have direct implications for ful reactive oxygen species, on phototoxic- sitivity, with the commonly used HeLa cell the design and implementation of live-cell ity. They were also interested in whether line being robust relative to the other tested super-resolution microscopy experiments, direct interaction of light with cells can lines. and they can also be used to guide the cause damage, which often goes unmen- The team also tested whether wavelength design of improved tools. Sauer believes that tioned. “We thought somebody should has a role in photodamage. They compared improved far-red dyes for super-resolution shed light on these obvious discrepancies several wavelengths of light at a range of microscopy and better tools for site-specific and remove the uncertainty,” recalls Sauer. light intensities and found striking results. labeling of proteins in live cells will be nec- The team systematically studied the They discovered that near-UV light is essary for live-cell imaging at the nanoscale effects of different light treatments on cells extremely phototoxic, even at low intensi- in the future. grown in culture. In one experiment, they ties, whereas far-red light causes minimal Rita Strack treated cells grown under different condi- phototoxicity, even at high intensities. A RESEARCH PAPERS tions with a range of light intensities. A day surprising result of this analysis was the Wäldchen, S. et al. Light-induced cell damage in later, they examined the effects of the light finding that a small change in wavelength live-cell super-resolution microscopy. Sci. Rep. 5, exposure by quantifying the numbers of can have a large effect on phototoxicity. 15348 (2015).

NATURE METHODS | VOL.12 NO.12 | DECEMBER 2015 | 1111