QSTORM-AO September 2019 Phone Meeting

September 24, 2019

by Carol Lynn Alpert

Participating:  Jessica, Peter, Abhijit, Abby, Faiz, Yixiao, Carol Lynn.

News:  Abby’s paper accepted to the Journal of Chemical Physics!   

Next Milestones:  Test both DNA-caged QDot micelles and our new QD-DNA origami structures using the “strand invasion” technique in a flow cytometry set-up on the STORM scope with HepG2 cells.  Complete incoherent holography set-up with microscope.

Report:  Optics Team – Georgia

In his post this month Abhijit describes the continuing effort to obtain good STORM images of the myopodia in neuromuscular junctions, using Alexa dyes.  They’ve found they have to stain multiple times to achieve higher labeling density in the samples: take 20,000 images, then restain, and do it again.  They’ve engineered a way to do this while the sample is on the microscope.  They’ve also been working at reducing the amount of excess background noise caused by scattered laser light when performing incoherent holography at low photon counts. They separated the excitation and emission paths and switched to an objective lens with a smaller collection angle.  This helped a bit, but not as much as they had hoped.  More work to be done….

Report:  QDot Team – Ohio

After having solved the DNA melting point problem by designing and ordering new DNA, Faiz and Abby found that the new DNA is giving them trouble conjugating to the QDot, a problem they had previously solved with the old DNA. They now have to go back through every step of the click chemistry process to figure out where it’s going wrong.  In addition, they discovered they had inadvertently chosen a cell line for the immunochemistry reaction that was incompatible with the antibody.  They are now switching to human HepG2 cells and will repeat all the controls with the new cell line.  Catch up on the details here.

Report:  Communications Team – Boston

With help from our newest member, Faiz Kahn, we made a new QDot Team member page entry.  We are pursuing background research for the film, and came across a nice 2018 superresolution review article by Xiawei Zhang and collaborators. (Sigal et al., Science 361, 880–887). We continue to be inspired by the perseverance of our QSTORM collaborators as they patiently work through each of the biological, chemical, engineering, and optical challenges that arise.  We’re keeping our fingers crossed that we will be able to film a QSTORM breakthrough this winter with both research teams.

To Do’s:

All: Please check for errors in this post and alert the author.  Please post any new publications, posters, talks in the Pubs section.

QDot Team: Conjugate AuNPs to the new Azo-dsDNA sequence (the one with with the correct melting points) and test FRET photoswitching capability with complementary QDot-azo-dsDNA conjugates.  Switch to new cell line, HepG2, to match current primary and secondary antibodies.   Confirm high specific antigen binding ratios.  Make caged coumarin dye samples and PC3 QDots.  Faiz to repeat conjugation steps using prior DNA sequences to verify procedure before returning to conjugation with the new DNA sequence that has the correct melting points.

Optics Team:  Order flow channel imaging chamber slides to use under STORM objective.  Obtain HepG2 cell line.  Finish light sheet design.  Submit paper on imaging 100 nm polystyrene beads with holography.

Communications Team:  Write and post September summary blog and solicit corrections from teams.  Pursue film R&D. Interview collaborator Daicho Kamiyama and research assistant Melissa Inal on their research with neuromuscular junctions in fruit flies.

Our next meeting is set for Thursday October 10, at 1 pm.