January 23, 2018
by Carol Lynn Alpert
Our first meeting of the new year! All onboard but Jessica who, sadly, was attending the funeral of a colleague.
Abby and Kil Ho entered the QSTORM blogosphere this week! But first we heard from Abhijit, who titled his post "First Light." And here it is: a 200 nm fluorescent bead imaged through the newly completed astigmatic path of the microscope.
This assembly of pixels is the diffraction pattern picked up from the glowing bead, with a tiny square indicating the localization point and the yellow line indicating the full width, 15 pixels, of the light intensity data plotted below. (The peak of the curve indicates the localization point.)
Each pixel is 86.6 nm square, and the "Full-Width Half Maximum" of the Gaussian curve shown below was measured at 318.53 nm, using a 1.4 Numerical Aperture oil objective. Not bad, considering you want your measurement as narrow as possible, and the absolute physical limit for a Point Spread Function is 300 nm. Peter and Abhijit are within 5% of the limit!
This is a great step forward, but Peter and Abhijit were not happy with the amount of drift produced by the new Smaract stage. It needs to be as stable as possible, given that the goal is to get 20 nm resolution of multiple fluorescing QDs. But the Smaract stage drifted 120 nm over a 30-minute period during this trial. Even in a ten minute period, during which 10,000 frames were captured, the stage drifted the equivalent of a few pixels. With trouble-shooting and phone calls with the Smaract company and videos sent back and forth, the problem has been narrowed down. The stage actuators for the Y axis are not working as they should. Ideally, Peter and Abhijit want to limit drift to 1 nm. Their previous microscope could only limit it to 80 nm., and they had hoped that this stage would at least get them under 10 nm. Of course, other variables can affect stability - building vibration, air currents, but they want to be sure they don't have a faulty stage.
Meanwhile, the QDot team reported progress on both the DNA Embedding and the Loops, Trains, Tails approaches.
Kil Ho has had success transferring the Ocean Nano red CdSe/ZnS core/shell combos from organic (oily) to aqueous (water) solution, while also preserving their absorbance and fluorescence peak. He is currently seeking to add additional ZnS layers. He began by optimizing the ligand exchange procedure by increasing the amount of 3-MPA (pH 10) and the ligand/QD mixing time. Small aliquots were taken during the ligand exchange process and the particle stability was examined by centrifugation (low speed). If the particles were not stable, they formed a pallet. Next, instead of applying the ZnS coating in a closed glass vial with a high temperature controller, he did it in a beaker on a hotplate. (Previous trials had suggested the “under-shooting” is better than “over-shooting.")
Kil Ho used a UV-Vis spectrometer to examine the absorbance of QDs before and after coating the surface with the additional ZnS layers. (Fig. 2) He confirmed that the additional ZnS layers did not cause a shift in wavelength; however, overall absorbance increased. He plans further studies to see if he UV-Vis spectrometer results can help determine the number of ZnS layers added.
Kil Ho also used Transmission Electron Microscopy (TEM) to examine QD size before and after adding additional ZnS coating. (Fig 3)
Figure 3 (C) shows the normalized size distribution of QDs based on the ImageJ analysis. The average sizes of QDs in both cases were not statistically different than the average Feret's length of approximately 6.8 nm; however, it was noted that the size distribution of QDs after additional ZnS coating showed broader range with the largest QD size ~ 11 nm.
To ensure the success of the ZnS coating procedure, Kil Ho is currently aiming to coat QDs with excess Zn. If that approach works, he expects to see a dramatic increase in the size of the QDs. Once he can confirm robust ZnS coating on the QD surfaces, he will begin to pursue DNA embedding. DNA strands will be added during the ZnS coating process.
Loops, Trains, Trails
Will the trials and tribulations of loops, trains, and trails, ever end?
When we last checked, Abby had found two divergent populations of Zn coated CdSe QDs emerging from her synthesis procedure. She went back to step 1, checked the reference paper, and decided to match their green QD Cd:Se core ratio of 1:10, rather than 1:2 ratio normally synthesized in the Winter lab. Despite good fluorescence yield as synthesized, the purification procedure did not produce consistent quantum yield cores, indicating a too-sensitive surface. And, the fluorescence peak wavelength was considerably blue-shifted, indicating incomplete growth (Fig 1, below). As a result, Abby was not able to test subsequent shell growth. Her future efforts will focus on producing robust cores followed by shell growth.
Biocompatible QDs and DNA conjugation:
Successful transfer of organic QDs in oily solution to biocompatible water solutions requires an efficient transfer of the organic ligands to pyridine in the first step, followed by ligand exchange with PC3 (Fig. 2)
The hot pyridine transfer showed excellent reproducibility for the commercial red CdSe/ZnS QDs. The following ligand exchange of pyridine to PC3 is triggered by incremental increase of the pH to over 10 using Tetramethylammonium Hydroxide (TMAOH). And, this time, with a fresh batch of TMAOH, the exchange was successful. Abby confirmed the results through absorption, fluorescence, long term storage and high speed centrifugation. The biocompatible PC3 coated QDs demonstrated successful transfer to low pH (pH 5) as well as physiological pH (pH 7) buffers that are involved in DNA conjugation chemistries. However, fluorescence decreased. Abby will pursue methods of maintaining a high quantum yield for the QDs.
Abby also further explored the EDC chemistry for DNA conjugation to these QDs. Her experimental batch displayed solubility at the high magnesium salt concentrations required for DNA origami conjugates, hinting towards successful conjugation. She will test these with DNA origami hinges for confirmation of the conjugation.
In Museum news, Karine made a post highlighted the contributions of the youngest new QSTORM team member, Sam from Weymouth Massachusetts. Sam, age 6, came to the Museum with his Mom, Meaghan Young, and they sat in on Karine's "Making Molecular Movies with QSTORM" museum presentation. Sam's illustration, shows many highlights from the show, including Jessica's Quantum Dots turning on and off, the molecular "machine" that hauls cargo along the microtubules inside cells, the nervous system, and the view under the microscope.
We'll meet again February 21st.