Understanding how a protein moves around a cell helps researchers understand the protein's function and the cellular mechanisms for making and processing proteins. This information also helps researchers study disease, which at a cellular level may mean that a protein is malfunctioning, stops being made, or is sent to the wrong part of the cell. But nanoparticle probes that are too big can disrupt a protein's normal activities.
Now a team of scientists led by Bruce Cohen of Lawrence Berkeley National Laboratory's Molecular Foundry, a U.S. Department of Energy (DOE) nanoscience center, has figured out how to grow light-emitting nanocrystals small enough to not disrupt cell activity but bright enough to be imaged one at a time. The results were publöished in ACS Nano.
"Scientists have been trying for years to study protein behavior by tagging them with light-emitting probes," said Cohen. "But the problem is finding the right kind of probe. Our approach is to make upconverting-nanoparticle probes small enough that they shouldn't disrupt protein behavior."
The Foundry team wanted to avoid both blinking and bleaching, so they turned to nanocrystals of sodium yttrium fluoride (NaYF4) with trace amounts of lanthanide elements ytterbium and erbium, which, they discovered, emit bright, steady light ideal for bioimaging. More importantly, these nanocrystals "upconvert" light, absorbing low energy photons and re-emitting them at higher energies. The advantage of upconverting nanocrystals is that cells don't upconvert light themselves.
"The other advantage to upconverting nanocrystals is that near-infrared light is a lot less damaging to cells than, say, visible or ultraviolet light," said Cohen. "That means when we do these very long imaging experiments using intense powers of light to see single molecules, we're using wavelengths that are pretty benign to cells."
A Combinatorial Solution
Nanocrystals of NaYF4 can form in two different geometries called alpha and beta. The beta-phase nanocrystals are more efficient at upconversion and thus better for bioimaging, but they're also harder to grow.
Imaging & Microscopy Issue 4 , 2012 as free epaper or pdf download
In order to nail down the growth parameters to get reproducible beta-NaYF4 nanocrystals, the team used the Molecular Foundry's WANDA robot - the Workstation for Automated Nanomaterial Discovery and Analysis.
Smaller nanoparticles means less light, so the team had to find the sweet spot: How small could they make them and still be able to image individual nanocrystals in a live system? "That's one of the nice things about having this control is that we can not only make them down to, say, 5 nanometers, but we also know the conditions for making them bigger if we need to make them brighter," Cohen said.
To help understand the geometry of their nanocrystals, coauthor James Schuck asked a summer intern to make a computer model of the crystal structure. Andrew Mueller, a high school student from Vistamar School in Los Angeles, went well beyond a simple crystal structure though.
"I started out just putting shapes together based on what was in the literature for the crystal," said Mueller. "Then I wanted to show how it looked in a nanocrystal so I moved the camera around in the structure and panned out to show how atoms come together in a nanocrystal." Mueller later added animation of two photons being absorbed and upconverted to a single emitted photon.
Next, the team wants to put the upconverting nanocrystals into action and actually map single proteins moving through a cell. "One of the things we'd like to study is how two neurons come together, how two brain cells come together to form a synapse - the spaces between neurons responsible for all brain activity," Cohen said. "It's known that there are certain pairs of proteins that come together from two neurons and they find each other and form a synapse but the question is, how many of those do you need? How many pairs of proteins? Is just one interaction enough to cause a synapse to form, do they reverse themselves, and so forth? Now that we know how to make exactly the nanoparticles we want, the next step is to test them in a cell."
Alexis D. Ostrowski, Emory M. Chan, Daniel J. Gargas, Elan M. Katz, Gang Han, P. James Schuck, Delia J. Milliron, and Bruce E. Cohen: Controlled Synthesis and Single-Particle Imaging of Bright, Sub-10 nm Lanthanide-Doped Upconverting Nanocrystals, ACS Nano, 2012, 6 (3), pp 2686-2692, DOI: 10.1021/nn3000737
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