The Preparation and Handling of Hydrated and Liquid Samples for In Situ Imaging Using TEM

Protochips manufactures the Poseidon system to enable in situ imaging of liquid and hydrated samples in the transmission electron microscope (TEM).

Liquid is separated from the vacuum between two E-chips, which serve as semiconductor specimen supports and loaded into the tip of the Poseidon TEM holder.

A transparent silicon nitride (SiN) membrane is combined into each E-chip. One E-chip contains an integrated spacer layer as indicated in yellow in Figure 1. The liquid thickness is set by this layer which enables the chamber to be easily constructed for flow or static operation. The second E-chip is much larger than the other and is flat. It is pressed against the spacer E-chip to form the sample chamber.

Figure 1. E-chip configuration

Several materials and equipment are provided to aid as a guide for making the E-chips and basic Poseidon samples. These include stereoscope, carbon fiber tweezers, well plate or spot dish, hot plate, filter paper, beakers or dishes, HPLC grade solvent, dry-pump station, pipet (0.5-10µL), residue-free compressed air, eyelash tools, alcian blue, and glow discharge or plasma cleaner.

Handling and Cleaning of E-chips

E-chips are offered in ‘gel- paks’ with the membrane side of the chips facing upwards direction. Poseidon E-chips comprise a very thin SiN membrane and therefore has to be handled with care to avoid breakage.

To remove the E-chips from the gel-pak, the chip edges have to be gripped with the carbon fiber to prevent the silicon frame of the E-chip, from damage.

E-chip Drying

To avoid contamination and residues from dust particles while drying the E-chips, residue-free compressed air can be used. Primarily, the edges of the chip’s edges have to be gripped using carbon fiber tweezers and removed from the final rinse.

Using a filter paper, the excess liquid can be wicked off by touching the chip’s edge. Next, air should be directly aimed across its surface until it is dries as it is being held by the carbon fiber tweezers.

E-chip Cleaning

E-chips have to be prepared in a clean environment, and high purity, HPLC-grade solvents have to be used to prevent particles from settling on the surface of the E- chips during and after the cleaning procedure.

First, dip the E-chips in a dish or beaker of acetone and agitate them by shaking the beaker for a few minutes. Then put the E-chips into a dish containing methanol and shake for 1-2 minutes. It has to be ensured that the E-chips don’t become dry during the transfer.

Surface Preparation of E-chips

To improve liquid flow and aid sample adherence to the E-chip surface, the SiN surface of the Poseidon E-chip can be modified prior to the experiment.

Hydrophobic Surface

The E-chips can be heated on a hot plate to dry out residual moisture from the surface, and thereby increasing its hydrophobicity. This type of treatment enhances adherence of non-polar materials such as the lipid to the SiN surface.

Hydrophilic Surface

While working with polar or aqueous solvents, a hydrophobic surface can obstruct establishing flow through the chamber, therefore, the surface of the E-chip has to be made hydrophilic prior to working with samples in polar or aqueous solvents. This can be easily realized by establishing a positive or negative charge to the E-chip surface.

As an alternative to plasma cleaning or glow discharge, the surface can be made hydrophilic by dipping the E-chips in a cationic copper phalocyanine dye, alcian blue. To establish a negatively charged surface, the E-chip has to be plasma cleaned or exposed to a glow discharge. This treatment also assists in removing hydrocarbon residue, which can be present on the E-chip.

Sample Loading

Figure 2. Loading the Poseidon holder

Many methods can be used to load the samples into the Poseidon holder (Figure 2). Tip assembly and sample loading can be done under a stereoscope having a 10x magnification. Prior to loading the chips into the holder, prime the lines with solvent or water so as to ensure correct sealing and to maintain hydration.

The excess liquid from the E- chip can be removed by dabbing its sides with a filter paper. After the E-chips are placed, close the lid using the pin provided at the front of the tip and lock it with the three brass screws.

Suspended Samples

Suspended samples, such as virus, bacteria, nanoparticles, proteins, and macromolecules can be applied using a small volume pipette (0.5-10µL).

The E-chip can be applied with certain samples, which subsequently can be dried and stored for imaging later. Samples have to be resistant to drying artifacts, free from aggregates and sufficiently dilute to avoid formation of salt crusts, which can cause erroneous seating of the two E-chips.

Such samples will have to be rehydrated by coating a drop of solvent or buffer between the E-chips during flow. This treatment method will also prime the sample chamber for setting up flow.

Viscous and Adherent Cell Samples

Thick or viscous samples such as gels, emulsions, creams, and oils can be imaged in an undiluted state using a static cell composition. It is possible to directly grow adherent cells on a flat E-chip using basic tissue culture conditions.

Moreover, fiducial markers can be placed onto the E-chip’s surface by coating gold nanoparticles on the chip prior to sample loading.

Conclusion

All the materials and equipment defined above act as a guide for preparing E-chips and basic Poseidon samples. This handling and surface preparation guide will assist in situ imaging of liquid and hydrated sample analysis in TEM.

About Protochips

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.

This information has been sourced, reviewed and adapted from materials provided by Protochips.

For more information on this source, please visit Protochips.

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