Minutes of the March 26th Phone Meeting

CL-icon-small-2Karine-icon-smThis expanded phone meeting included all PIs, post-doc Jianquan Xu, and graduate students from three labs: Qirui Fan from Ohio State, Nati Chen from Brown, and Kayvan Tehrani from Georgia. The student participation marked the growing inclusiveness of our collaborative research effort and the increasingly important contributions made by the students engaged in it.

Cheers   We shared a virtual glass of champagne over the journal Nanotechnology’s acceptance of the micelle quantum dot imaging paper, with its multiple QSTORM authors. Thanks to Qirui, Jessica, Peter, and Jianquan for managing the additional down-to-the-wire data collection and anaylsis.

Kudos for QSTORM   Carol Lynn reported on her conversations with the PI of our IBIV Ideas Lab funding program, Eduardo Rosa-Molinar, and original NSF Program Officer, Steve Ellis. We learned that QSTORM is well-regarded as a “poster child” for some of the most successful aspects of the Ideas Lab funding mechanism. The team is perceived to have carried out a truly interdisciplinary, coordinated collaborative research program which has significantly impacted the research directions of all PIs and their students, and our post-doc, Jianquan. Carol Lynn is developing a proposal about the Ideas Lab approach to building innovative collaborations. Steve Ellis is still involved, since he has been asked to participate in an upcoming Ideas Lab focused on innovative undergraduate teaching in biology. NSF is thinking about designing a side-by-side program in which results from the Ideas Lab approach could be compared with results from the typical individual investigator approach in the same research area. Beth suggested that the role the MOS team has played in coordinating communications and managing the website has been significant in keeping us focused and on track and building a growing sense of community among PIs and students. (Thanks, Beth!) Ge commented that other IBIV Ideas Lab groups he’s worked with have suffered from a lack of organization, so their collaborative efforts have tended to peter out. However, at least a few of the other participants are continuing to work together.

Accelerating Publication    Since we have had a few conference papers, are soon to publish our first mainstream paper, and have a couple in the pipeline, all bodes well for continuing the collaborative effort. But to do so without a gap in funding would require pressing ahead faster with the papers in progress. The team asked Ge if he could use Peter’s data in the revision resubmittal of his Tau paper. There had been the expectation that Peter was going to be a co-author of this paper. The team also asked Ge if he could get out Jianquan’s paper on cell penetrating peptides in an upcoming conference proceedings, rather than holding back until his group finishes the new experiments exploring particle size as a major variable. Jessica suggested EMBC 2014, the 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, which meets in Chicago in August, and Ge agreed this was a good idea. He tasked Jianquan with preparing a conference paper submittal, which is due April 7. This is good for Jianquan, who will then have a second paper out of this project before the Post-Doc funding is finished. And the full research paper, including particle size experiments with polystyrene beads, can go into a high-impact journal a little later. The polystyrene beads, in sizes ranging from 20 nm to 100 nm are expected to arrive next week. They will be carboxylated and coated with streptavidin before being used with the cell penetrating peptides.

Rabbit Muscle     Meanwhile, Nati, Jianquan, Kayvan, and Peter have been collaborating over the best protocols to use to prepare new rabbit psoas fiber samples for STORM imaging. The first set, though beautiful, where deemed too dim in the green channel for STORM imaging (see images and succeeding discussion at http://www.qstorm.org/2014/03/19/imaging-muscle-fibers-phalloidina488-alpha-actinina680/ Nati has now prepared a new set of samples using a revised protocol and has booked confocal microscope time for later in the day. If all goes well, these will be on their way to Georgia within a day or two. Everyone decided that the confocal image could serve as an effective preliminary screening device, even though it is tuned to the 633 channel and the Alexa dye is 680. Nati’s samples include both fixed, relaxed, single-stained muscle fibers (alpha actinin/Alexa 647); and fixed, not-relaxed, double-stained muscle fibers (alpha actinin/Alexa 647 and phalloidin/Alexa 488). Peter thinks that there may be a technical issue with relaxant – since the signal was very bright before Nati started relaxing the muscle fibers. Last time buffers proved to be a problem with STORM, Jianquan got around it by finished the sample preparation in the Georgia lab and imaging immediately.

Microtubules and Non-Specific Binding   Jianquan reported that the Georgia lab is getting good STORM imaging of microtubules with the 565 Q Dots. The 655 Q Dots have some issues with non-specific binding that may be resolved with improvements to protocol. Jianquan, using commercial 655 QDs prepares control batches that don’t contain the primary antibody in order to determine where the problems lie. However, both batches had too much non-specific binding. Kayvan used Qirui’s 655 QDs to stain microtubules, and saw too much non-specific binding at high concentration. The trick is to get concentration right – too dilute, and there is not enough luminescence; too high and there is too much non-specific binding. They’ve tried a range. While they could see the microtubules at intermediate concentrations, there was not enough signal to get good STORM images. Kayvan mentioned that he has some nice Adaptive Optics algorithm work in STORM images but hasn’t posted yet to the blog.

Optimizing Antibody and Particle Loading   Jessica suspects that the optimal concentration issue may be resolved by calculations that quantify the precise amount of antibody to load. She and Qirui are going to try to calibrate that, setting out first to increase the antibody loading of particles, first optimizing conjugation with streptavidin, then trying with the IgG antibody. Up to now, they’ve been getting less than 10% of IgG antibody loaded onto the QDs.

“Making Molecular Movies with QSTORM” Debuts at COSI Columbus Karine shipped clones of the QSTORM dopplegangers to Columbus and gave two very well received presentations during COSI’s NanoDays event. The 2pm show in particular had a large crowd and many audience members came up to Jessica after the show to chat and ask questions. Most heartwarming: “How can I become a scientist like you?”  Qirui was a pro with the QD and gold nanoparticle quenching demo, introducing quantum dots and how they can be used in microscope imaging. He found visitors to be very interested in quantum dots and their applications. Best of all, two staff members at COSI learned the demo and COSI intends to adopt it into their repertoire of stage presentations. MOS provided COSI with all the props, slides, script, and video, which are also available to other museums through the NISE Network catalog, at http://www.nisenet.org/catalog/programs/making_molecular_movies_qstorm QSTORM will also be featured at the Museum of Science’s NanoDays event on Saturday, April 5th in Boston. And then, in conjunction with the QSTORM research meeting in Pittsburgh, QSTORM members will be at the Carnegie Science Center in Pittsburgh, and Karine will present “Making Molecular Movies” twice. Ge and possibly Jessica will join Karine on the stage for a Q&A following the presentation.

Speaking of Pittsburgh… Ge confirmed that everyone has been able to book a hotel room in the block he set up. Everyone flying has made their reservations. Ge will send out a draft agenda shortly.

We fit all this discussion in one hour!

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