October 11, 2018
by Carol Lynn Alpert
All present but Abby, stuck in the lab….. We missed you Abby! But a new grad student has joined the QDot team: Faiz Khan. Welcome Faiz!
Abhijit posted two updates this week covering the last couple of months of work. The team’s malfunctioning laser is back and working fine again. The collaboration with Daichi Kamiyama is proceeding. Now that they’ve succeeded in optimizing the labeling process with microtubules, they’ve turned their attention to the real object of study, neuromuscular junctions in fruit flies. The pair in Fig. 4 show a widefield and then STORM reconstructed image of the filopedia of a neuron, the extension by which neurons reach out and migrate toward a particular destination. Those slender fingers are barely 100 nm in diameter and the resolution of the image is within 10 nm!
The central challenge the team faces is “read noise,” emanating from internal reflections in the lenses of the objective and/or perhaps fluorescence in the glass. (They have sealed the whole set-up in a light tight box, so external light is not the problem.) In his Oct 10 blog post, “It might be too noisy,” Abhijit provides examples of the effects of read noise on localization, and graphs the calculations, showing that with the burden of an irreducible read noise of .5 electrons, the photon output will have to exceed 5,000 per 10 millisecond image capture. The fluorescent beads they’ve been using as samples are not bright enough. On this phone call, they asked Jessica’s team to provide QDots in the 670-690 range (the range they’re currently tuned to). On a cover slip, these will give them 35-45 nm fluorescent imaging targets. Jessica’s team said, “yes.”
Kil Ho unfortunately wasn’t able to make much progress this month as his suppliers were having purification difficulties and postponed shipments several times. The batch just arrived on Monday, so he’s plunging in…
Abby was stuck in the lab and Jessica walked us through her Oct 9 Blog Post. First, she needed to confirm that the DNA had successfully conjugated to the QDots and was conformed in a way that left one tail outward, available for binding to the complementary gold NP-DNA required for quenching. She did this using gel electrophoresis purification, commonly used in protein separation. This proved more effective than the previous column separation method, since it separates based on both size and charge. She was able to confirm conjugation. She tested proper conformation by introducing DNA origami hinges and performing gel electrophoresis to determine if the QD-ssDNA conjugates were binding to the hinges at the desired places. They were. Finally, she imaged the QD – hinge composites, confirming everything was as it should be (Fig. 6) Abby is now ready to begin synthesizing photoswitchable QD-DNA-AuNP conjugates for use in STORM imaging.
The team at MOS has launched an effort to develop a 3D animation of the holographic light sheet STORM-AO process the Optics Team is developing. Megan Litwhiler and Karine Thate had a conference call with Peter last week, to help distinguish and understand the new imaging technologies Peter and Abhijit are combining in their quest to reach unprecedented resolution in thick tissue. So far as we know, they are the first team to try to combine STORM with AO, plus light-sheet, plus holographic imaging techniques. We’ve not yet been able to find very good existing graphic resources, so we may have to start from scratch. This is a call out to Peter and Abhijit to help us locate anything that would be helpful.
The MOS team posted their new film, “The 2018 Quantum Matters™ Science Communication Competition Finals” at www.mos.org/qmc2018 It’s a fun film and has already been a hit at NSF. We premiered it at the Center for Integrated Quantum Materials annual meeting last week, and were asked by NSF NNI director Mike Roco to bring it to the 2018 Nanoscale Science and Engineering Directors Meeting in Alexandria in December.
Our next meeting will be November 8th, at 1 pm.