QSTORM-AO November 2018 Phone Meeting

November 8, 2018

by Carol Lynn Alpert

All present but Abby, out on a job interview.  

Optics Team

The Optics team is moving their imaging lab and office to a new space on campus, so they had to disassemble their microscope.  No new work to report. But Abhijit is preparing a paper to present on their current work at Photonics West in early February.

Fig. 1  Left: Disassembled lab space.  Right: New space, floor all shiny and awaiting equipment delivery.

The Optics Team is also awaiting delivery of a batch of QDots in the 670-690 range, to match the laser they’re using, and which they’ve asked the QDot Team to procure.  They are hoping that the brighter fluorophores will give them enough signal to drown out the irreducible “read noise” they’ve been picking up when imaging weaker fluorescent beads. These are big QDots and will provide 35-45 nm fluorescent imaging targets. However, it turns out that their large size makes them tricky to synthesize, and Kil Ho has been able to find only one supplier in the U.S., a company he has not ordered from previously.  (There’s also one company in Europe and one in China). The 680’s are on order, but not yet delivered; probably because they are being synthesized on spec.  Jessica says that it’s challenging, because as the wavelength increases, it becomes more difficult to achieve  quantum confinement in QDot form and bulk material characteristics emerge. (The difficulty of trying to optimize this synthesis is one of the reasons the QDot lab does not want to get sidetracked from their green QD focus.)

Q-Dot Team

Kil Ho and Abby authored a poster on both their QD-DNA conjugation methods for the 2018 American Society of Chemical Engineering annual meeting, and invited their undergraduate mentee Thomas Porter to present it in Pittsburgh last week.  We hope they upload it here soon.

Kil Ho guided us through his new blog post, “Toward DNA-embedded Quantum Dots: Take VI,” posted November 7.  In the post, Kil Ho reviews his progress achieving single strand (ss) DNA conjugation during the process of accreting additional zinc sulfide (ZnS) shell layers around the QD core. He adds free Zn ions and thiol (-SH)-modified ssDNA into a solution containing QDs at an elevated temperature (90 °C).  Previous results seemed promising; a red shift in the fluorescence emission indicated enlarged QD core/shell combos. This month, to confirm the results, he repeated the embedding procedure, attaching cyanine dye molecules (Cy5) to the ssDNA.  Since the QDs emit green fluorescence (550 nm) and the Cy5 emits red fluorescence (~660 nm); the presence of both red and green signals would confirm successful DNA embedding. However, the results were disappointing. While the QD emission peak shifted from 550 to 560, indicating some additional ZnS shell layering, the amount of Cy5 emission at 660 was negligible; indicating that the ssDNA had failed to embed in any meaningful way.  Kil Ho will try to repeat the experiment with more rigorous calibration and may also try a different approach; for example, using a saltier solvent solution.

Jessica walked us through Abby’s Nov. 6 post, “Azobenzene and the actuation system.”  Since Abby has already perfected the “Loops, Trains, Tails” method of conjugating ssDNA to QDs and ssDNA to gold nanoparticles (AuNPs), she is ready to begin testing photoswitchable QD-azoDNA-AuNP conjugates for use in STORM imaging.  That switching system depends on moving a gold nanoparticle closer to or farther from the QD through changing the conformation of an ssDNA linker treated with azobenzene molecules.  (See Fig 2 below.) In the presence of visible light, the two single DNA strands want to “hybridize,” forming the classic double helix structure, which brings the gold nanoparticle (AuNP) close enough to the QD to “quench” its fluorescence by sucking off a little of its Forster Resonance Energy.  The azobenzene molecules, in their “trans” conformation help bind the two ssDNAs into the double helix. However, in the presence of ultraviolet (UV) light, the azobenzene molecule goes into its "cis" conformation and retreats to just a single ssDNA, causing the DNA double helix to separate once again into single strands.  This “dehybridization” releases the quenching AuNP from the QD and allows the QD to resume emitting light.  Thus, the cycling between UV and visible light triggers the cycling between QD-on and QD-off states, producing just the type of selective emissions essential to STORM imaging.

Fig. 2 The QD-azoDNA-AuNP reversible photoswitching mechanism.

Abby is currently investigating methods of achieving higher yields of the QD-azoDNA-AuNP conjugations, through improved purification techniques.  But she also decided to go ahead and do a simple test of the current optical setup for actuation and detection of photoswitchable azoDNA, using neither QD nor AuNPs.  She did this by substituting the dye Cy5 for the QD, and a dye-quenching molecule (Q) for the AuNP. There was good news: When the Cy5-azoDNA-Q went into the solution of Cy5-ssDNA, the dye’s peak fluorescence emission dropped by 80%, confirming significant quenching. (See Fig 3.)

Fig. 3  Once the Cy5-azoDNA-Q pair formed, the dye molecule was quenched.

But, when Abby tried to reactivate the Cy5, by applying UV light for five-ten minutes, it did not resume fluorescing.  To troubleshoot, she will have to confirm the strength of the UV light source and possibly try adding more azobenzene molecules into the ssDNA conjugation process.  Qirui, one of the original members of the QDot team, had been able to achieve both quenching and re-fluorescence a few years ago using the azobenzene DNA hybridization and dehybridization scheme, so we are fairly confident we'll be able to fix this.

Communications Team

Carol Lynn reported that the MOS team has been able to get a story on super-resolution imaging into the Museum’s annual Top Ten Science Stories of the Year presentation that will be delivered dozens of times to hundreds of Museum visitors in December and January.  The story focuses on Nobel Prize winning microscopist Eric Betzig’s recent publication (Science  20 Apr 2018: Vol. 360, Issue 6386, eaaq1392) of a new technique combining lateral light sheet structural imaging (a different type of superresolution technique) with adaptive optics.  Betzig’s group made stunning videos of activities going on inside live embryonic zebrafish cells, included in the Figures & Data section of the paper (See https://doi.org/10.1126/science.aaq1392.)  We will use the story to hint at the much superior resolution QSTORM-AO will achieve (at lower cost) by using STORM with light sheet, adaptive optics, and holography as well as switchable quantum dots, once all our work comes together.

CL also commented that it looks promising that NSF will be able to provide funds to help make the 2019 Quantum Matters™ Science Communication Competition national in scope.  Folks on the call had positive comments about the film, which is posted at www.mos.org/qmc2018  

Our next meeting is scheduled for noon on December 12.