June 19, 2019
by Carol Lynn Alpert
Participating: Jessica, Peter, Abhijit, Abby, Faiz, Carol Lynn
Kil Ho is off practicing for his thesis defense which is tomorrow, Jun 20. Good luck, Kil Ho!
This just in: Jianquan Xi, QSTORM’s former traveling post doc has been promoted to Assistant Professor at the University of Pittsburgh: One more solid accomplishment of our QSTORM journey. Jianquan came to the U.S. when we hired him as our PostDoc. He began in Jessica’s lab, rotated through Peter’s lab, and did his last stint in Pittsburgh with former QSTORM collaborator Ge Yang. Congratulations, Jianquan!
Optics Team – Georgia
Abhijit and Peter are preparing a manuscript for publication on their success using of holography in imaging 100 nm polystyrene beads despite low photon counts. As we were speaking, Abhijit was looking at just-arrived packages containing all the new materials needed to set up two-color imaging on the STORM microscope. He’s also continued working with biologist Daichi Kamiyama to explore high-resolution imaging of neuromuscular junctions in fruitflies. Last month, they were able to achieve good detail of the growth cone of a nerve cell attaching to muscle tissue at depth inside a full embryo. But they are having trouble isolating and imaging the filopodia of the muscle cells. The stain is not selective, and so the entire muscle cell lights up, making it impossible to isolate and localize individual fluorophores on the filopedia. This was not a problem with the nerve cells; because they are much smaller there was less background noise from the non-selective staining. Peter commented that the trouble they're having with muscle cells is just one more incentive to get the light sheet technique working.
QDot Team - Ohio
With Kil Ho graduating, Faiz and Abby are focusing more on the remaining hurdles to achieving stochastic QDot labels for STORM imaging based on a QD-DNA-AuNP conjugate reversible photoswitching mechanism.
They’ve been able to assemble these QDot-DNA-gold nanoparticle conjugates and to quench the QDots by bringing the gold NP closer to the QDots, but they have not yet been able to “unquench” the QDots. It turns out that the “melting point” of DNA - the temperature at which double-stranded helix hybridizes into separate single strand DNA - is a key factor in determining whether both quenching and unquenching can occur, and this melting point is highly variable, based on factors such as the length of the DNA strands, the base pair composition of the strands, the concentration of azobenzene molecules (azo) attached to them and so on. So Faiz and Abby will focus on designing new DNA – azo composites that have melting points that allow them stability in the hybridized double strand structure with the azo linker molecule in its trans form at room temperature (quenching mode), but are unstable in the dehybridized single strand structure with the azo linker in its cis form (unquenching mode). They summarize the goal this way: “For an efficient photoswitch, we require a DNA sequence which has a cis melting point below room temperature and a trans melting point well above room temperature.” They also note that a previous study indicates that incorporating an equal number of azo moieties on both sides of double-stranded DNA structure can help. So Abby and Faiz will insert azo on both of the complementary single strands they design. See the full report here.
Meanwhile, Jessica’s NIH grant proposal for continued work on the cage method of QDot quenching is pending, and scores are high. She has a provisional patent, and two companies have expressed interest, but she would like to further refine the technique. It will be useful for cancer diagnostics, but it may also be useful for imaging, and on this call she asked if Peter would like to test them with STORM imaging. He agreed to let her know what type of QDot marker label she should prepare for him to test imaging in a fixed cell.
Communications Team - Boston
Carol Lynn is in the process of defining the scope of the QSTORM film, and she had a number of broad picture questions for Jessica and Peter. Here is the discussion regarding one of them:
Question: Is the idea of enhancing super-resolution imaging with QDot fluorophors now more widely accepted than when QSTORM began?
Answers: There’s still interest in using them since they would greatly increase imaging resolution, but applying them has been more challenging than previously expected, for a number of reasons: a.) Commercial QDots have not proved very stable – or uniform. b.) Failure often occurs during the aqueous transfer required for biological applications. c.) Partly because of (a) and (b) above, people often publish one-time results that they and others find difficult to reproduce. d.) Our project introduces further difficulty with the necessity to (1) conjugate QDots to biomolecules and figure out how to get the right number of molecules on the surfaces, and (2) engineer the quenching mechanism necessary for STORM imaging. Furthermore f.) It will be particularly difficult to get the conjugated QDots inside living cells to image molecular structures, because the conjugates are currently so large. On the other hand, QDots have now proved their value for labeling biomarkers on the surfaces of cells for diagnostic purposes. They are an order of magnitude brighter than dyes currently in use.. And eventually, the QDot reliability issue and the other challenges will be solved and imaging with them will be realized.
Our next meeting is set for July 16 at 11 am.