(303c) A Platform for Retaining Native Morphology At Sub-Second Time Scales in Cryo-TEM

Authors: 
Croote, D., Brown University
Godfrin, M., Quest Consultants Inc.
Bose, A., University of Rhode Island
Tripathi, A., Brown University
Lee, J., Sungkyunkwan University



Introduction

Cryogenic transmission electron microscopy
(cryo-TEM) is a widely used tool for morphological analysis in biology,
chemistry, and polymer science. Using this technique, samples can be imaged in
their native state without fixing or staining, which often cause fundamental conformational
changes. Sample preparation for cryo-TEM involves
removing the excess liquid deposited on a cryo-TEM grid by blotting with filter
paper, which can be seen in Figure 1c-d, and plunging the grid into a cryogen
(usually liquid ethane), resulting in vitrification. The blotting step is
required to thin the sample, allowing the electron beam to pass through the
sample, and to ensure complete sample vitrification. During this process,
however, native sample morphology is compromised due to high shear rates
inflicted on the sample. Additionally, species may non-specifically adsorb to
the filter paper, resulting in a non-native sample concentration when imaged.

Platform
Overview

aiche.pngWe report the development of a new sample
preparation device based on capillary action that overcomes the above
limitations. The use of a glass removal capillary aligned parallel to the grid,
as shown in Figure 1a-b, results in reduced shear and lower absorption of
particulate material from the sample. Furthermore, a deposition capillary
aligned perpendicular to the grid coupled with LabView-controlled hardware
allows for precise and sub-second resolution for time resolved studies. We
demonstrate each of the features of this platform using model samples, and
where appropriate, compare our results to those prepared using current
vitrification platforms.

Results

The efficacy of our new sample preparation technique
is shown through the imaging of CTAB in phenol, Ultra Downy fabric softener,
calcium carbonate intermediates, and a carbon black nanoparticle
suspension. A mixture of 50 mM CTAB and 50 mM phenol, which forms wormlike micelles in aqueous
solution, was utilized in order to compare the morphological differences
resulting from choice of sample preparation method. The natural morphology of
the wormlike micelles exhibits an entangled structure, which was captured after
preparing a sample with our method. A sample prepared using a Vitrobot system with blotting and without relaxation
results in wormlike micelles that are oriented parallel to each other, aligned
in the direction of flow. Numerical studies were used to quantify the shear
causing this alignment; it was found that shear during blotting is one to three
orders of magnitude greater than for our method of parallel capillary action. To
illustrate the time resolution of the system, the evolution of a mixture of aqueous
calcium chloride and sodium carbonate from amorphous calcium carbonate at 700
milliseconds to calcium calcite at 5 was depicted. Another advantage of our
system is the capability of imaging higher viscosity fluids. Cryo-TEM images
were successfully taken of Downy fabric softener with a viscosity ~100x higher
than that of water and a surface tension lower than that of water. Lastly, images
of carbon black particle suspension prepared using our method showed species
adsorption was reduced by ~20x compared to blotting.

Conclusion

We have developed a novel cryo-TEM sample
preparation technique that allows for the vitrification of samples without the
shear induced orientation of artifacts that are typical of the conventional
cryo-TEM blotting sample preparation. Through the use of a glass removal
capillary, we have also effectively eradicated the issue of the altering of
sample nature through the adsorption of sample species typical of current preparation
methods. Our technique also allows for sub-second time resolution, which is beneficial
for the imaging of the intermediates of chemical or biological processes
present at sub-second intervals. Our results confirm that this new sample
vitrification device opens up previously unattainable regimes for sample
preparation and imaging and is a powerful new tool for cryogenic transmission
electron microscopy.

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