November 6, 2019
by Carol Lynn Alpert
Participating: Jessica, Peter, Abhijit, Faiz, Elizabeth, Carol Lynn
Current Goals: (1) Apply the “strand-invasion technique” to DNA-caged QDot micelles and to compact uncaged QDot-DNA conjugates in a flow cell set-up on the STORM scope with U87 cells; also continue to pursue the original FRET reversible photoswitching using azobenzene. (2) Complete light sheet illumination set-up with the microscope. (3) Explore the holographic technique again, using QDots from NN Labs. (4) Collaborate with neurobiologist to test out STORM improvements.
Report: Optics Team – Georgia
The parts for the light-sheet illumination add-on to the STORM scope have arrived, and Peter and Abhijit have begun assembling it. The paper submitted to Optics Letters received favorable reviews, and Peter and Abhijit are submitting a revision today.
In his post this month Abhijit demonstrates with step-by-step illustrations how the team manages to correct for the lateral chromatic shift that frequently occurs when practicing two-color imaging. In Fig. 4 from his post, you can clearly see the impact on paired images before (A) and after (B) chromatic shift correction has been applied. In the post-correction image (B), the green-tagged synapsin can be seen integrated with the red-tagged growth cone. It is this synaptic conjunction that is the target of the investigations of Kner lab neurobiology collaborator Daichi Kamayama.
Report: QDot Team – Ohio
Faiz, who is learning to master the QDot conjugation process, was able to get over last month’s temporary setback getting water solubility with NN Labs QDots. He did it by replacing the contaminated reagent inadvertently used. He was then able to successfully transfer the QDots from organic to aqueous medium, and to begin focusing on the second step: conjugating the ssDNA molecules to QDots through surface carbodiimide chemistry. That was also successful. He verified that he been able to attach about 5 ssDNA per QDot, but there is some question about whether that measurement can be compared to one made by Abhilasha, which recorded an average of .5 per QDot. Jessica pointed out that having more ssDNA per QDot is probably OK for the cage method, but not for the reversible QD-DNA-AuNP conjugate photoswitching strategy using azobenzene; it would best to have about 1 ssDNA per QDot to avoid too much aggregation.
In the meantime, Elizabeth achieved success conjugating the complementary ssDNA strands to the surfaces of the gold nanoparticles, and Thomas has prepped the azobenzene attachments. That means that future experiments can finally explore reversible photoswitching of the QD-AuNP-DNA nanocomposites with azobenzene - something we’ve been looking forward to for a long time! Congratulations team.
Elizabeth is also working on the alternative technique of DNA caging of QDots, with the cages reversibly attached to secondary antibodies. There had been too much non-specific binding using HepG2 cells, and her switch to U87 cells did not solve the problem. She fixed it by switching to a different buffer solution. She then discovered an excess of secondary antibodies in the system and switched purification methods to remove fix that problem. She is now trying to trouble-shoot issues comparing secondary antibody signals using cages vs. controls.
Report: Communications Team – Boston
In her blog post, Carol Lynn reports on her phone meeting with Kner lab collaborators Daichi Kamiyama and Melissa Inal in order to get a better understanding of their research into the neuromuscular junctions in fruit flies. In other news, the film of The 2019 Quantum Matters™ Science Communication Competition has been released, and is available for viewing at www.mos.org/qmc2019. Brown graduate student Sue Shi won first prize for her talk on developing quantum dots for solar power harvesting.
We discussed our goal to help Peter test the holography method again, using QDots to boost the signal strength. Last month we had decided that the Ohio lab would send antibody conjugated QDots, but in this meeting we decided that Peter would be better off beginning with non-conjugated red QDots ordered from NN Labs.
All: Please check for errors in this post and alert the author.
QDot Team: next steps:
1. For the effort to apply the strand invasion technique on PC3 treated compact QDots, Abby and Thomas need to conjugate them to an antibody-binding DNA and test in U87 cells.
2. For both that goal and to achieve the QDot caging method, Elizabeth will find a solution to the antibody problem.
3. For the azo reversible photoswitching method, Jessica and Faiz will verify amount of ssDNA conjugation per QDot and Faiz, Thomas, and Elizabeth will test them with the gold nanoparticles.
Optics Team: next steps:
1. Acquire a complete set of imaging data on the myopedia in neuromuscular junctions.
2. Complete and test light sheet add-on to the STORM setup.
3. Order 610 QDots from NN Labs and attempt holography in a particle tracking experiment (after adding the light sheet).
Our next meeting is set for Thursday, December 12, at 1:30.