Outcomes from Four Years of Collaboration and Innovation
Carol Lynn Alpert – July 1, 2015

The QSTORM team’s long-term goal has been to provide an enhanced super-resolution imaging technique that can probe deeply into the molecular-scale activities of biological systems. Existing super-resolution techniques have been limited by the short-lived, low-luminance capacity of fluorescent dyes and by optical distortions induced by imaging through depth into cell tissues. The QSTORM team has sought to harness brighter, longer-lasting quantum dots in place of fluorescent dyes, and to introduce adaptive optics algorithms to correct for optical aberrations in thick tissues.

Adaptive Optics and QSTORM

The Kner Lab made considerable progress in both these areas:

• Applying a genetic-algorithm machine-learning technique with a novel evaluation metric to quickly hone in on an optimal wavefront correction strategy for any particular sample. A four-fold increase in point-localization precision was demonstrated at a depth of 50 microns in Drosophila central nervous system tissue. See the paper here.

• Applying a “blueing” technique with QDots to achieve two-color, three-dimensional QSTORM imaging of microtubules and mitochondria, with 24 nm lateral and 37 nm axial resolution. This breakthrough makes it possible to harness the superior luminescence of QDots for STORM imaging, while efforts to develop photoswitchable QDots for STORM proceed in parallel. See the paper here.

Quantum Dots for STORM

The Winter Lab made progress synthesizing water-soluble micelle-coated quantum dots with enhanced biocompatibility, luminance, and longevity. They were also able to modulate fluorescence intensity to some extent using a secondary laser pulse. See the paper here.
• The micelles could be packed with multiple and multicolor QDots (as well as “MagDots”), useful for diagnostics as well as imaging.
• Be tagged with targeting molecules to label particular structures inside cells.
• Be used for STORM imaging through the spectrum shifting “blueing” technique (see Kner Lab section above).

The quest to produce photoswitchable  QDots to make them more useful for STORM imaging has proven more elusive, despite some promising (and patented) results modulating QD fluorescence using gold nanoparticle quenching agents connected to target molecules by photo-activated azobenzene-DNA linkers. This work continues.

Molecular Targeting for QSTORM

The Brainerd Lab and the Yang Lab found insurmountable difficulties using microinjection techniques to introduce targeted QDots into living cells. Regardless, both labs were able to further the QSTORM effort and their biological investigations.

• The Brainerd Lab, which started out probing live zebrafish embryos, discovered an excellent alternative model for investigating molecular scale muscle dynamics: glycerinated rabbit psoas muscle, which maintains its ability to contract even when fixed. The lab made progress imaging muscle fiber components using immunostaining with STORM, TEM, and confocal microscopy and provided labeled samples for the Kner Lab.
• The Yang Lab, in collaboration with the Kner and Winter Labs, developed a successful strategy for introducing QDots into mammalian cells using a cell penetrating peptide (CPP) approach and published this result.
• The Yang Lab was successful in getting a new STORM system installed at Carnegie-Mellon and has been making new discoveries on the distribution and function of the tau protein in axonal nerve transport. The group also continues to make progress in various computational aspects of STORM imaging for studying intracellular transport. (See publication and conference presentation list)

Training and Advancement

• Three of the four non-tenured university PI’s achieved tenure promotions and multiple awards and honors during the course of the project. The tenured PI, Beth Brainerd was elected President of the International Society of Vertebrate Morphology. Ge Yang and Peter Kner received NSF CAREER awards. Jessica Winter started a company and received eight innovation and teaching awards.
• Rotating Post-Doc Jianquan Xu published two STORM papers, became a father and is now in a full-time research position at the University of Pittsburgh Cancer Institute.
• Winter Lab Post-Doc Gang Ruan is now a full professor at Nanjing University.
• 16 graduate and undergraduate students were actively involved in QSTORM research, 9 of them authoring or co-authoring QSTORM publications or conference presentations, and 9 of them obtaining degrees or new jobs. In a confidential survey [Jan-Feb 2015], 80% of the students said that their participation in the project greatly influenced their subsequent education, training, and career choices. Most often cited was their involvement in the multi-lab, multi-disciplinary research effort, and 93% reported that it made them more interested in pursuing collaborative research further.

Public Impact

• 6,500 museum visitors have experienced the 20-minute “Making Molecular Movies with QSTORM” museum presentation through June 30, 2015. A “legacy version” continues to be popular. Visitor evaluations show substantial engagement and learning. The presentation has been “packaged” and made available for replication to other science museums through the NSF Nanoscale Informal Science Education Network. It has been featured at the MRS Fall Meeting, COSI Columbus, and the the Carnegie Science Center, Pittsburgh.
Video of the museum presentation and podcasts with Jessica Winter, Peter Kner, and Ge Yang are posted on the QSTORM playlist on the popular NanoNerds YouTube channel, which has over 1200 subscribers. Also posted there are two animations about the project produced by students at the New England Institute of Art.
• The QSTORM website (qstorm.org) has received more than 35,000 unique visitors as of July 1, 2015, with over 8,500 first time visitors in each of the last two full years (2013-2014). The site has hundreds of blog posts, and dozens of pages of faculty and student bios, lab logs, images, background, interactive visual timelines of the history of biological imaging and of the progress of QSTORM research, plus links to publications, and media.
• The MOS team gained significant capacity to report on and interpret biological and super-resolution imaging research for broader audiences, and to enhance the effectiveness of multi-disciplinary, multi-university  collaborative research teams.