July 4, 2018
by Carol Lynn Alpert
All hands were in attendance yesterday; today there will be fireworks. The big news is that Jessica and her beau Aaron are getting married. Congratulations you two!
Abhijit had previously reported testing the new “incoherent holography” 3D imaging technique on the new STORM microscope to image 1 micron microspheres loaded with yellow/green fluorescent dye. This month, with the help of a better performing camera and a laser, he successfully imaged 200 nm microspheres loaded with red fluorescent dye. The figures in his blog post, reproduced below, show three holographic images he engineered, by setting the mirrors and beam-splitter in three varying patterns to produce interference phases. Each image shows faint roughly circular diffraction rings surrounding each bead’s position. The final reconstructed image resolves the 9 upper microbeads into sharply distinct light-emitting particles.
Abhijit will run further tests, to see if there are other configurations of the optics that can produce even better results in reconstruction. Once he’s found on optimal configuration he will arm the system with a higher quality camera. He wants to be able to go down to 100 nm beads next. (The goal is to resolve down to 20 nm, to single molecules.) Abhijit also needs to be able to reduce the exposure time from the current 500 milliseconds to 20-50 milliseconds. All the more reason to max out the number of photons being emitted, eventually by getting those dye particles replaced with QDots.
Kil Ho spent most of the month at Argonne National Labs working on another project. He also landed an interview with Dow Chemical and received a job offer post-Ph.D. Just before Kil Ho left for Chicago, he had persuaded the lab’s current QDot supplier to agree to replace any batches they send that turn out not to behave as they’re supposed to. This is a result of the previous work he did (see the May 23 Phone Meeting Report) carefully documenting quality control issues among the suppliers. The variability was playing havoc with his experiments. The fresh batch they sent, however, arrived just before his trip to Argonne. He will have to pick up from there.
Abby posted a well-organized summary of her progress toward conjugating QDots (QDs) with single-stranded DNA. She is having success with the first step: transferring the QDs to a water solution using the phytochelatin-3 (PC3) ligand method (also known as “loops, trains, tails”). Since the next step, conjugation to DNA in that aqueous solution, had been problematic – and DNA is expensive - she had performed the conjugation chemistry trials using a dye group as a DNA substitute. Having achieved that, she resumed using the same chemistry to conjugate the DNA, but this failed, presumably because of non-specific interactions of DNA with the QD-PC3 surface. Two possible solutions are being investigated: (1) modifying the DNA and (2) modifying the QD-PC3 surface to reduce its interactivity. To modify the QD-PC3 surface, Abby’s ordered a batch of polyethylene glycol (PEG) groups to partially passivate some of the charged surface ligands. To modify the DNA, she’s ordered some single-stranded DNA consisting only of thymine. (A literature review showed that - of the four DNA nucleotides -thymine demonstrates the least interaction with nanoparticles.) Of course, changing the DNA sequence also changes its molecular weight and size, so that means Abby has to re-engineer the size-separation columns she uses to purify the QD-PC3-DNA conjugates from excess/unreacted DNA. She faced two options, either using a new gel or a longer column. She decided to go with the more economic option, the new gel. Unfortunately, this gel is back-ordered at the supplier, so we won’t know the results for weeks.
In the meantime, Abby and Thomas, an undergraduate research associate, began scouring the literature to search for alternative conjugation chemistries in case this one (carboiimide chemistry) fails, despite all the modifications being tested. Thomas suggested “Click chemistry” for a new direction. Although relatively new, this crosslinking chemistry has demonstrated better conjugation efficiencies. So Abby and Thomas will open a new front on the campaign to conjugate DNA and QDots.
Then, in the middle of the call, Jessica sent out some slides showing schematics of “DNA cages” she’s using on another project to surround dye-bearing micelles. Just earlier, while warming her lunch, she had had a hunch that perhaps these structures could also provide an alternative route to achieving the stochastic (randomly varying) photon supply required for STORM. The cages slowly detach from the cell surface through complementary DNA interactions, gradually releasing dye molecules. Jessica will send a batch to Peter so he can test them with the holographic STORM technique, using them to label a cell surface protein. It will be somewhat like using the “blueing” technique Peter tested several years ago, but in reverse – the number of fluorophores would slowly increase over time rather than decrease, but still allow discrete sets of localizations.
In other news, the evening following this July 3rd phone meeting I viewed the July 1 Last Week Tonight with John Oliver segment on the topic of genetic engineering, in which he uses scenes from several films, including Larry Klein’s recent NOVA, to provide a cautionary tale about responsible use of biotechnology. (Larry is our QSTORM filmmaker, and I’d recently reported in this blog the broadcast of this documentary, Can We Make Life.) See the new post for Oliver's take on the subject.
We’ve scheduled our next phone meeting for August 3 at 11 am. Peter suggests a Skype or Google Hang-outs video call.