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Biology, Energy, Materials Science, Technology

Bright future for protein nanoprobes

Surprising new rules for creating ultra-bright light-emitting crystals that are less than 10 nanometers in diameter have been discovered by a team of researchers. These ultra-tiny but ultra-bright nanoprobes should be a big asset for biological imaging, especially deep-tissue optical imaging of neurons in the brain.

Researchers at Berkeley Lab’s Molecular Foundry created upconverting nanoparticles (UCNPs) from nanocrystals of sodium yttrium fluoride (NaYF4) doped with ytterbium and erbium that can be safely used to image single proteins in a cell without disrupting the protein’s activity. Credit: Andrew Mueller

Researchers at Berkeley Lab’s Molecular Foundry created upconverting nanoparticles (UCNPs) from nanocrystals of sodium yttrium fluoride (NaYF4) doped with ytterbium and erbium that can be safely used to image single proteins in a cell without disrupting the protein’s activity.
Credit: Andrew Mueller

The term a “brighter future” might be a cliché, but in the case of ultra-small probes for lighting up individual proteins, it is now most appropriate. Researchers at the U.S. Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) have discovered surprising new rules for creating ultra-bright light-emitting crystals that are less than 10 nanometers in diameter. These ultra-tiny but ultra-bright nanoprobes should be a big asset for biological imaging, especially deep-tissue optical imaging of neurons in the brain.

Proteins are one of the fundamental building blocks of biology. The cells that make up tissues and organs are constructed from assemblies of proteins interacting with other biomolecules, while other proteins control nearly every chemical process inside a cell. Studying the location, assembly, and movement of specific proteins is essential for understanding how cells function and what goes wrong in diseased cells. Scientists often study proteins within cells by labeling them with light-emitting probes, but finding probes that are bright enough for imaging but not so large as to disrupt the protein’s function has been a challenge. Fluorescent organic dye molecules and semiconductor quantum dots meet the size requirements but impose other limitations.

“Organic dyes and quantum dots will blink, meaning they randomly turn on and off, which is quite problematic for single-molecule imaging, and will photobleach, turn-off permanently, usually after less than 10 seconds under most imaging conditions,” Schuck says.

“Cells don’t naturally contain lanthanides, so they don’t upconvert light at all, which means we can image without any measurable background,” Cohen says. “And we can excite with near-infrared light, which is a lot less damaging to cells than visible or ultraviolet light. These are great properties, but to make our UCNPs more compatible with cellular imaging, we had to develop new synthetic methods to make them smaller.”

However, when Foundry scientists shrunk UCNP size, following the conventional design rules, they found that loss of brightness became a major issue. UCNPs smaller than 10 nanometers were no longer bright enough for single molecule imaging. This prompted the new study, which showed that factors known to increase brightness in bulk experiments lose importance at higher excitation powers and that, paradoxically, the brightest probes under single-molecule excitation are barely luminescent at the ensemble level.

“This discovery came about really as a consequence of the multidisciplinary collaborative environment at the Molecular Foundry,” says Daniel Gargas, co-lead author of the Nature Nanotechnology paper. “By utilizing our daily contact and friendships with scientists throughout the Foundry, we were able to perform highly advanced research on nanoscale materials that included the study of single-molecule photophysics, the ability to synthesize ultra-small upconverting nanocrystals of almost any composition, and the advanced modeling/simulation of UCNP optical properties. There aren’t many facilities in the world that can match this collaborative atmosphere with such high levels of scientific characterization.”

Chan’s computer models predict that the new rules are universal for lanthanide-doped nanocrystal hosts and he is now using the Foundry’s WANDA robot (Workstation for Automated Nanomaterial Discovery and Analysis), which he developed along with co-author Delia Milliron, to create and screen for the best UCNP compositions based on different operation/application considerations and criteria.

In the course of discovering the new rules for designing ultra-small UCNPs, the research team also discovered that complex levels of heterogeneity exist within the emission spectra of these UCNPs. This suggests that emissions from the UCNPs may be originating from only a small subset of the total emitters.

“Future studies may determine how to engineer particles consisting of only these super-emitters resulting in even brighter emissions from ultra-small UCNPs,” Gargas says.

Reference: Daniel J. Gargas, Emory M. Chan, Alexis D. Ostrowski, Shaul Aloni, M. Virginia P. Altoe, Edward S. Barnard, Babak Sanii, Jeffrey J. Urban, Delia J. Milliron, Bruce E. Cohen, P. James Schuck. Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging. Nature Nanotechnology, 2014; DOI: 10.1038/nnano.2014.29

 

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