QSTORM-AO March Phone Meeting

March 30, 2018

by Carol Lynn Alpert

Not all hands, not this time. Peter announced that Abhijit was off getting married.  Congratulations Ahbijit!  Nevertheless, he did manage to send in a lab report, and Peter guided us through it.  It has not yet been posted in the Optics Team blog, but we’ll cut Abhijit some slack.

Optics Team

Alas, the issues with the Smaract stage are not yet resolved.  You may recall that it was not holding position well, drifting hundreds on nanometers on one axis.  It was shipped back to Germany, but the Smaract engineers could find no problem and shipped it back.  Now it has a new issue – a clicking/jerking movement.  Trans-Atlantic trouble-shooting is ongoing.

In the meantime, Abhijit set about testing phase masks to use on the Spatial Light Modulator (SLM), resulting in the beautiful images below (Fig 1).  The SLM takes the place of using two mirrors to modulate the beam of light coming from the sample, producing interference patterns used for holographic imaging.  The phase masks replicate the form the wavefront would take if the interference was being produced by two mirrors. 

Phase Masks
Fig. 1  Wave interference patterns caused by application of 3 different phase masks.

The Optics team has also been collaborating with Dr. Rabindranath De la Fuente from the Veterinary school to perform STORM imaging of mouse oocytes.  His goal is to determine the arrangement of chromatin in the nucleosomes. These are relatively thick samples (~60 -80 um), so there is a considerable amount of background noise.  The team has been trying to create a sheet of light to reduce the background noise, and one of the ideas was to place a micro-prism beside the sample to create a sheet of light inside the sample. They were successful in mounting the prism on the cover-glass bottom dish but it was apparently a messy affair involving epoxy glue.

QDot Team

When we last checked in, Abby, who is pursuing the “Loops, Trains, Tails” method, had succeeded in optimizing aqueous transfer of QDots, but they were not yet conjugating with DNA.  She decided to do a control experiment to see if they would conjugate with dye, using the same EDC-sulfo NHS chemistry.  They did, confirming the conjugation chemistry is operational. However, Abby had used a very high dye:QD ratio, 3500:1.  Since DNA is much more expensive than dye (5 micromoles, enough for just two experiments, costs about $1000), she tried the conjugation chemistry using a lower ratio of DNA: QD, 200:1.  That failed.  (See Fig. 2)

180330 conjugation data
Fig. 2  DNA conjugation succeeded with dye, but not with QDots. 

Next, Abby will try boosting the DNA:QD ratio by substituting in some cheaper amine-terminated “filler” molecules for some of the amine-terminated DNA. These may help stabilize the reaction.   Abby learned one other important thing during these experiments: using column purification rather than centrifugal purification achieved better retention of successful dye:QD conjugation, so she will adopt that procedure with the next batch of DNA as well.  See Abby’s March 29 post for more details.

Kil Ho is still troubleshooting the DNA conjugation-during-shell growth alternative method to DNA:QD conjugation.  He had already determined that the presence of ZnS shell growth, but he had been unable to confirm DNA embedding in the shell.  He tried by testing binding to DNA hinges. The results were inconclusive, and the DNA hinges he was working with were a bit old, so he wanted to try to verify the DNA embedding using fluorescence microscopy with collaborator Josh.  The results seem to show that DNA embedding was at least partially successful, but not good enough.  Plus there was some aggregation.  So, he too is going to have to play around with the DNA:QD ratio. 

Fig. 3 Commercial QDots are expensive; the lab will optimize their own synthesis
and production techniquesfor conjugation with DNA

In the meantime, Kil Ho is also working on replacing commercially-manufactured NN-Labs CdSe quantum dots with homegrown lab QDs.  (See Fig. 3) After getting in a fresh batch of chemicals, synthesis of green CdSe QDs worked very well, and they were successfully transferred to water through the ligand exchange method; however, they did not fluoresce after the exchange. The next trouble-shooting steps include checking once more with green-core commercial QDs, as the previous batch had been red.

Up Next:  PIs will meet on April 19 to go over drafts of our Annual Reports.  NanoDays is next week at the Museum of Science!