This article describes an experiment that involves imaging of rotavirus particles in solution with a Poseidon™ in situ liquid TEM holder. The high resolution, 3D reconstructions of biological assemblies from individual particles within the liquid can be evaluated using the images obtained from the experimental findings.
The experiment allowed observation of unique structural subpopulations of particles in liquid versus the homogeneous structural states of ice-embedded and chemically fixed samples.
The experiments were carried out in Deborah Kelly’s lab at the Virginia Tech Carilion Research Institute. A lipid monolayer (Affinity Capture) containing polyclonal antibodies was allowed to functionalize one Poseidon E-chip™ surface against VP6, a rotavirus protein.
The functionalized E-chip was then applied with a solution consisting of 0.1mg/ml rotavirus and a low concentration of contrast reagent. As the amount of staining reagent was not enough to fix the rotavirus particles, and the reagent was included to improve the contrast for downstream image processing.
Using an FEI Spirit BioTwin TEM with a tungsten filament that operates at 120kV under low dose condition, the images of the rotavirus particles in 150nm liquid layer were recorded. Following this, 3D reconstructions of both in situ and vitreous ice embedded specimens were produced using the RELION software. Figure 1 shows the projection averages of the in situ and frozen specimens.
Figure 1. Projection averages of rotavirus particles calculated from (A) in situ liquid and (B) frozen specimens. Contrast of the ice averages (B) is inverted for ease of comparison.
Results and Discussion
Figure 2 shows the single 3D volume average with 25Å resolution calculated from a population of 600 particles. The diagram indicates that the most of the rotavirus particles were divided into two subpopulations: marked in pink (23%) and blue (65%).
Two additional reconstructions, however, included only two additional reconstructions i.e. yellow (5%) and grey (7%) of the total particles in the image stack.
Figure 2. Single 3D volume average with a resolution of 25Å was computed from a population of 600 particles
Therefore, the observation of four distinct subpopulations of the particles suggest some degree of structural heterogeneity as the particles were tethered to the liquid chamber surface and not fixed.
The image stack of cryo-TEM prepared samples showed a single, statistically significant population against the observation for the in situ specimens.
Poseidon allows the user to attain nanometer to atomic resolution imaging of samples under dynamic liquid conditions, and hence it is compatible with different materials and biological samples. It is applicable for both TEM and STEM, and also analysis using correlative light and electron microscopy (CLEM) .
The integration of consumable, Poseidon E-chip devices used for producing the sample chamber into tissue culture and standard sterilization techniques enable direct cell growth on the surface of E-chip, facilitating nanoparticle uptake analyses, cellular imaging and labeling.
The Poseidon TEM holder is provided with two or three liquid ports and thus ensuring easy configuration for static, flow or mixing operation. It maintains a hydrated environment and enables observation of dynamic processes including particle-particle interactions, nucleation, nanoparticle growth and self-assembly.
The results show the first example of a 3D reconstruction of biological species in their native liquid environment. In addition, using traditional cryo-TEM imaging, a ~25Å resolution was obtained from averaging 600 rotavirus particles compared to ~24Å resolution of the same sample.
When imaged in solution instead of vitrified ice, a greater degree of structural heterogeneity was observed among virus particles. The dynamic subpopulations or heterogeneity may be a result of Brownian or beam-induced motion.
Tethering particles to the E-chip surface using Affinity Capture allows visualization of the dynamic sub-states that cannot be observed while immobilizing particles through freezing or chemical fixation.
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Using its proprietary technology, Protochips is addressing the market need by transforming the most widely used tools in nanotechnology – electron and optical microscopes - from cameras into complete nano-scale laboratories.
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