Winter Lab Research Update

Gang Ruan at Histochemistry 2012
While Jessica was recovering from surgery, her colleague Gang Ruan presented the Winter lab’s QSTORM research efforts at Histochemistry 2012, held at the Marine Biological Laboratory in Woods Hole, Massachusetts. In this talk, Gang focuses on the effort to design the switchable quantum dots required for STORM super-resolution imaging.



Karine and Carol Lynn teamed up to write this summary of the Research Update Presentation delivered by Jessica Winter at the Athens, Georgia Meeting on December 11, 2011.

JESSICA WINTER’S LAB: Photo-Switchable Quantum Dots

The QSTORM goal for Jessica Winter’s lab at Ohio State-Columbus is to develop photoswitchable quantum dot systems optimized for STORM imaging in vivo. The term “photoswitchable” means that their fluorescence can be turned on and off by an external beam of light. If Jessica is successful, STORM imaging could reach new levels of resolution, enough to allow imaging of the molecular machinery at work inside living cells. QDs, brighter and longer-lasting than conventional organic dyes, would replace them as the fluorophores of choice in biological imaging. In 2008 Stefan Hell’s group published a paper demonstrating the use of QDs for another kind of super-resolution imaging (STED), with switching controlled by the modulation of red and blue lasers. Jessica intends to make a better QD system and a better modulation system, which will enable higher resolution STORM imaging at smaller scales.

Jessica’s team includes Gang Ruan, Qirui Fan, Kalpesh, and new Post-Doc student Jianqian Xu. The team’s first goal was to learn how to produce the type of QD that Stefan Hell’s group uses, because they want to improve upon them.

Manganese-Doped Quantum Dot Synthesis
“Hell’s particles” are manganese-doped zinc-selenium (ZnSe) quantum dots. Typical QDs – like the Invitrogen variety – are made using cadmium – and cadmium is highly toxic and is known to cause cancer. The reason Jessica is so interested in Hell’s QDs is that they are non-toxic. (Zinc and selenium are even in vitamin pills!) As a further advantage, the Mn-doped ZnSe QDs also have a great photon yield (emitting 30-40% of the photons they absorb). They emit yellow light when activated with a blue laser, and they can be modulated or partially quenched by a red laser.

Jessica’s colleague Gang Ruan first worked on the synthesis of the Mn-doped ZnSe QDs while working as a post-doc in Nie Shuming’s lab at Emory. They tried out several methods until they achieved versions with the same characteristics and photon yields reported by Hell. Jessica walked us through the steps of the synthesis, which was rather like watching a complicated dinner preparation.

Adding Micelle Coatings
Their next step with the Mn-doped QDs is to make them water-soluble. Hell used a set of ligands surrounding the QDs to achieve solubility; Jessica is keen on encapsulating small groups of the QDs within “micelle coatings.” These are formed from amphiphilic block copolymers. These are large macromolecules made of chains of smaller molecular units. They are hydrophilic – water soluble – on one end and hydrophobic – insoluble – on the other. They self-assemble into rings around the QDs, with the hydrophilic ends facing outward, forming a water soluble shell around the QDs). Jessica’s hypothesis is that these micelle coatings will protect the QDs from “photobleaching.” (Photobleaching is the scourge of microscopists because it dims the fluorescence.)  The micelle coating should therefore increase the longevity of the bright fluorescence emitted by the QDs and thus enhance their usefulness in microscopy.

Jessica’s team has succeeded in adding micelle coatings around small groups of conventional Invitrogen CdSe QDs. They sent these to Peter, who succeeded in demonstrating that they deliver a much better photon yield and for a longer period of time than the uncoated variety. Jessica’s team is just about to complete their synthesis of micelle-coated Mn-doped QDs – the non-toxic QDs from Hell’s lab. They’re very eager to collaborate with Peter to test out these new coated QDs for STORM imaging, and especially to compare their stability and brightness with uncoated QDs. Everyone will be very excited if they succeed in triumphing over Hell’s original particles.

Exploring Photoswitching Techniques
The Winter lab is also testing several methods of switching quantum dots on and off by applying a second source of light, most likely UV. (The first source is used to activate the QDs, and it is that activation, or excitation, that causes them to begin fluorescing, or releasing photons.)   Some of the most promising switching methods involve first conjugating (joining) a gold nanoparticle (NP) to a QD with a linker molecule. A gold NP in close proximity to an excited QD “steals away” the excitation energy that would otherwise be released from the QD in the form of photons, thus “quenching” the QD, or “keeping it in the dark.” But if that gold NP linker is broken or “cleaved” in some way – usually by a form of light, such as UV light -the QD can be freed of its overbearing quench-master NP and can begin again to shine. Why do we need to put the QDs through this routine of switching on and off? Remember that diffraction-busting, super-resolution STORM imaging relies on the microscopist’s ability to record the precise location of just a small fraction of fluorophores (photon-emitting sources) at any given instant. Whereas, if all the QDs were emitting light at the same time, all we’ll get is a big bad blur. This quenching process is known as FRET, as in “Don’t fret dear, your Fluorescence Resonance Energy Transfer has dimmed the lights again…”

Single-Use Gold Nanoparticle Quencher – Jessica’s team began with the modest goal of using a photocleavable linker to attach a gold nanoparticle FRET agent to the QD.   A flash of the right kind of light, and the link is severed and the gold NP floats away, and, voila! The QD resumes releasing its excitation energy in the form of photons.
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However, since the gold NP assembly does not then return and reattach, this is a one-shot deal – pouf! – the light goes on, the gold particle moves off, the QD begins to fluoresce – but then there is no way to reattach the cleaved linker and quench the fluorensce again. (Peter had a thought that maybe, if the photocleaving light trigger is applied in tiny pulses, and the rate of photocleavage varies, he might be able to use this in the same way he would use the bluing technique – which is also one-way.) But we’re all still rooting for Jessica to perfect the next technique, the REVERSIBLE QUENCH.

Reversible Gold Nanoparticle Quenching – The idea here is to find a gold nanoparticle linker that can not only be cued to separate the gold NP away from the QD in response to an external light trigger,  but could also then bring the gold NP back close to the QD, quenching it again, repeating this in and out movement again and again.
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The linker would stretch or shrink depending on an external trigger, and the variability in distance would be enough to Quench or Not To Quench. (Kind of like having a pull cord on a light switch going on and off.) Jessica’s group and Peter’s group are very excited about this, but the biologists in the room had some reservations. The trigger light is generally in the UV part of the spectrum, and living things don’t like a lot of UV light, but Jessica said the amount of UV needed would be tiny. She is eagerly awaiting delivery of a the linker components. She also anticipates having to make a molecular “tether” of some sort, to ensure that the gold NP linker doesn’t stretch too far away to be able to spring back to its close-in position with the QD.

Quenching through Photocleavable PEG Coatings – This technique does not involve gold NPs or FRET.  It is simply a QD coating made of PEG (polyethelene glycol) that falls apart when hit with the triggering beam of light.  The loss of the PEG coating makes the QD susceptible to photobleaching, sort of analogous to the “blueing” technique Hell uses.  Some very generous folks at NC State who shall remain anonymous because I don’t know their names – offered Jessica the use of some of their custom-designed PEG QD coating material, which she can use to try this.

Now, What About Doing It With Micelle-Coated QDs?
Jessica is very fond of her micelle-coated QDs, and, naturally, her mind is racing ahead to the issues that might be encountered when she tries to attach photocleavable – or photostretchable – linkers to them. And then there are also the issues that will come up when the QDs are also decorated with the organic molecules they will need to conjugate with their imaging targets inside cells. None of this is going to be easy.

Next steps for the Winter Lab

  • Complete the micelle encapsulation of Hell’s particles (the Mn-doped ZnSe QDs)
  • Work toward a paper with Peter’s group comparing the photon yield of micelle coated QDs and plain QDs, using both CdSe and doped-ZnSe varieties.
  • Work on bioconjugation of COOH and biotin groups with micelle-coated QDs (these will be critical to the immunohistochemistry involved in tagging intra-cellular molecules.
  • Continue to try to optimize single-use and reversible FRET-based linker technology.
  • Personnel: Gang is transitioning off QSTORM. He passed on his synthesis expertise with Hell’s particles to Qirui who is now a second year student. Qirui is passing that knowledge on to Jianqian and turning his attention to the FRET modulation techniques. Jianqian will master synthesis and manipulation of Hell’s particles and then do a rotation to Peter’s lab in Athens.
  • Peter is still planning to continue to try to optimize the bluing technique, but he is also eager to move on to the FRET techniques with gold nanoparticle conjugates and the PEG cleaving technique, once Jessica has functionalized them.