QSTORM-AO August Phone Meeting

August 3, 2018

by Carol Lynn Alpert

All hands were in attendance today, noting that summer is passing too quickly!  We discussed our timeline, given that Abby and Kil Ho will be finishing up their doctoral program next summer, and our instrument development grant from NSF terminates in April.  Peter and Carol Lynn will request a no-cost extension:  Abhijit will be working on the holographic STORM development at least through May 2020, and Carol Lynn's team has been reserving funds to begin film production when the Optics and Quantum Dot teams bring their parts of the technology together; hopefully, this coming spring.   Everyone reported good progress, so we are optimistic.

Optics Team: Tackling "Read Noise" with a New Camera

The holographic STORM instrument has been completed and the algorthms for processing the imaging feeds are working. Abhijit has been working steadily to increase the resolution of the system.  He has been successful imaging 1 micron and also 200 nm microspheres with a better quality sCMOS camera, but with exposure times of 500 milliseconds - too long for STORM-quality resolution.   He and Peter decided to go back to their simulations to try to determine how sensitive the "incoherent holography" technique might be to "read noise" at low light levels (500 photons).  "Read noise" is aberrations produced by factors such as camera electronics and low signal-to-noise ratios.  Peter and Abhijits' simulations showed that at low light levels, the system is indeed very sensitive to read noise. When read noise is greater than 0.1 e-, the incoherent holography technique cannot provide a good reconstruction. They switched over to use an EMCCD camera (iXon Life 897). Using the EM gain mode In the EMCCD camera, the read noise can be brought below 0.1 e- .  After making the necessary changes and finding the optimal camera conditions, they were able to image 200 nm red (580/605) fluorescent microspheres at 50 millisecond exposure times and successfully reconstruct the signal.  Figure 1 shows the three holographic images, and the reconstructed sharp image on the right. The jump down from 500 to 50 milliseconds is quite significant, and puts them into STORM range.  

The next steps are to image smaller beads (100 nm), and also to capture a 3D image stack while moving the bead within a 10um range, reconstructing the holograms at the best focus for each of the z-positions.  See more details and links to more information about "read noise: and EMCCD cameras at https://qstorm.org/node/486.

QDOT Team:  Breakthroughs!

Kil Ho: Toward DNA-Embedded Quantum Dots

Kil Ho has been wrestling for some time with variability in various batches of QDs from different suppliers and even from the same supplier (a paper on this common problem plaguing many researchers is forthcoming).  This time, applying some creative reasoning, he decided to see if there might be another route to make the aqueous transfer process more robust.  He substituted the organic solvent chloroform for the organic solvent hexane before proceeding to the aqueous transfer and ligand exchange steps of the procedure.  It worked!  Not only did the switch to chloroform solve the ligand exchange problem, it also addressed the prior failure of green QDs (too reactive because of their larger surface to volume ratio) to undergo successful transfer and ligand exchange.  In fact, following ligand exchange this time, the fluorescence intensity of the green QDs exceeded their base state!  Read all about it and see the images in Kil Ho's post.  Next, he will be addressing the variables in the conjugated DNA sequence with the goal of achieving a good binding match with the DNA linker that will bring the quenching gold nanoparticle in close proximity to the conjugated QD-DNA.

Abby: It Clicked!

Meanwhile, Abby has been assiduously testing multiple possible causes of the various failures of the "loops-trains-trails" method to get DNA conjugated to the surface of QDs. This month she investigated potential modifications to the DNA sequence as well as modifications to the QD surface groups.  These efforts were unsuccessful, although she has a few other troubleshooting ideas to check out.  The month's big breakthrough came when she applied a new technique called "Click Chemistry" to the conjugation task.  As she reports in this month's post, the fluorescence intensity results appear to confirm that conjugation occured. They showed not only the normal peak for the green QDs at about 610 nm, but also the 670 nm peak for the dye molecule attached to the DNA.  Now, at last, Abby can proceed to verification through conjugation with DNA origami hinges.

Communications Team

The team at MOS has been busy with science communication workshops for undergraduate interns and REU students, and with editing footage from the NanoDays and Quantum Matters(TM) Science Communication Competition presentations.  We spent some time coaching Kil Ho and Abby on their writing skills and are noticing improvements in blog post reporting from the doctoral candidates.   Carol Lynn has been invited to deliver a keynote at the 2018 Nanoscale Science and Engineering Grantees Meeting at NSF in December and Karine will deliver a panel member talk as well.  Carol Lynn will also be giving a talk at the 2018 NSF Science Technology Centers Directors Meeting at Berkeley in late August.  Our filmmaker Larry Klein's 2016 NOVA mini-series Great Human Odyssey has been nominated for a Jackson Hole Science Media Award, and the recent NOVA Wonders mini-series - for which he produced a film on genetic engineering - has been nominated as well.

We will meet next on September 6, 2018, 10:30-11:30.  Lab report posts are due at noon on September 4.

Have a good August everyone!