This article discusses the imaging of rotavirus particles in solution with a Poseidon™ in situ TEM liquid sample holder.
The images acquired were employed in the calculation of the first high resolution, 3D reconstructions of biological assemblies from individual particles present entirely within liquid.
This setup allowed observing unique structural subpopulations of particles present in the liquid against the homogeneous structural states of chemically fixed and ice-embedded samples.
Applications of Poseidon™ in situ Liquid TEM Holder
Poseidon can be used with a variety of materials and biological samples, facilitating nanometer to atomic resolution imaging of samples in dynamic liquid environments.
The Poseidon platform is well-suited for both TEM and STEM as well as for correlative light and electron microscopy (CLEM) analyses.
The consumable, Poseidon E-chip devices used to create the sample chamber can be integrated into standard sterilization and tissue culture methods, enabling direct cell growth on the E-chip surface for nanoparticle uptake analyses, cellular imaging and labeling.
The Poseidon TEM holder is offered with either 2 or 3 liquid ports, enabling easy configuration on an experiment-by-experiment basis for mixing, flow, or static operation.
Besides maintaining a hydrated environment, the Poseidon TEM holder enables observing dynamic processes, such as self-assembly, nanoparticle growth, nucleation, and particle-particle interactions.
All analyses were performed at Deborah Kelly’s lab in the Virginia Tech Carilion Research Institute. A lipid monolayer (Affinity Capture) consisting of polyclonal antibodies was used to functionalize the surface of one Poseidon E- chip™ against VP6, which is a protein found in rotavirus.
A low concentration of contrast reagent (0.2%w/v uranyl formate) and a solution consisting of rotavirus (0.1mg ml-1) was applied to the functionalized E- chip.
The amount of staining reagent was inadequate to fix the rotavirus particles and was added to improve contrast for downstream image processing. An FEI Spirit BioTwin TEM integrated with a tungsten filament operating at 120 kV under low-dose conditions (~5 electrons/Å2 for each exposure) was used to image rotavirus particles present in a 150nm liquid layer (Header Image Panel A).
The RELION software was used to generate 3D reconstructions of both the in situ and vitreous ice embedded samples, from 600 and 572 particles, respectively. 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.
A single 3D volume average with a resolution of 25Å was computed from a population of 600 particles, and is depicted in purple color in Figure 2. Most of the rotavirus particles were categorized into two subpopulations (blue (65%) and pink (23%)).
Two additional reconstructions consisted of only 7% (grey) and 5% (yellow) of the total particles contained in the image stack.
Figure 2. A single 3D volume average with a resolution of 25Å was computed from a population of 600 particles.
These four different subpopulations of the particles reveal the existence of some degree of structural heterogeneity among them and tethering of the particles to the liquid chamber surface, meaning that they were not fixed.
As opposed to what was expected for the in situ specimens, the image stack of cryo-TEM prepared samples showed the presence of a single, statistically significant population.
The results show the first example of 3D reconstructions of biological samples in their native liquid environment. Imaging in solution showed a greater degree of structural heterogeneity among virus particles than in vitrified ice.
This heterogeneity or dynamic subpopulations may be due to Brownian or beam- induced motion. Tethering particles to the E-chip surface through Affinity Capture facilitates imaging of the dynamic sub-states, which cannot be observed in chemical fixation or freezing due to immobilization of particles.
Protochips, Inc. is a rapidly growing early-stage company focused on providing the world's leading materials and life sciences research breakthrough analytical tools for targeted research and development of nano-scale materials.
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.
Protochips' core competency lies in the application of semiconductor techniques to development of MEMS devices capable of providing heat, electrical, liquid and gas environments to samples in situ.
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