Nanotechnology is driving scientists to better comprehend the real-time dynamics and structure-property relationship of various materials and biological samples under liquid conditions. Such understanding is crucial for a wide range of applications involving, for example, nanoparticle synthesis, self-assembly processes, (bio) molecular interactions, and biological activity in cells. In-situ transmission electron microscopy (TEM) observations in the liquid-phase is expected to lead to better scientific understanding, the discovery of phenomena at the nanoscale in liquid not visible before, and results in novel and innovative applications. Here we present the development of the “Ocean System”, which is an easy-to-use add-on that enables in-situ liquid studies inside the TEM (Figure 1). It consists of an optimized TEM holder that uses a microfluidic chamber as sample carrier, replacing the traditional copper grid. Such device, referred to as Nano-Cell, acts as a multi-functional and micro-sized laboratory that keeps the sample in a fully hydrated state. Furthermore, the system includes an external test station that guarantees the safe loading of the holder into the TEM. Each Nano-Cell consists of two chips (Figure 2) that are sandwiched together to form a sealed microfluidic compartment. Both chips are covered with silicon nitride providing an electron transparent window and ensuring their chemical inertness and biocompatibility. Samples are prepared directly onto the electron transparent windows, which allow for the electron beam to pass through for in-situ imaging. Biological cells can also be directly grown on the chips.

In order to control the liquid thickness to improve imaging resolution, the experiment can be customized by selecting the best-suited spacer based on sample size. Having direct access to the electron transparent windows enables local functionalization of the membrane´s surface, empowering the user to further control the microfluidic environment. The holder tip contains a precision slot with various alignment poles that ensure self-alignment of the top and bottom chips. Similarly, it contains a by-pass structure that prevents overpressures during liquid handling, and that allows rapid liquid exchange in the tubing, since the flow cross-section in this channel is much larger than that of the liquid path between the chips. The tip closure mechanism uses alignment balls, so that the tip correctly closes when screws are tightened independently on the applied force. The mechanism prevents over-compression of the O-rings and ensures that no shear stress will be transferred to the Nano-Cell, as these could damage fragile samples (i.e. biological cell). Additionally, the modular design ensures reliable results with easy replacement of all holder parts, such as tubing, holder tip and the Nano-Cell (Figure 3). This is particularly important, as it prevents cross-contamination between different experiments, and the tubing can be easily replaced by the user if these become clogged. In addition, the tip can be rotated by 180°, so that depending if one wants to use TEM or STEM, the optimal resolution can be achieved for the sample, i.e. TEM achieves the highest resolution for objects below a liquid layer for a downward traveling electron beam, while the opposite is true for STEM.

The Ocean System can be used to study dynamic processes of nanoparticles. E.g. gold nanoparticles can be loosely attached to a SiN membrane. Their detachment during the experiment can be triggered by increasing the induced electron dose. This can provide useful information such as the interaction of nanoobjects (e.g. agglomeration, self-assembly, sintering) in different liquids. Figure 4 shows Au nanoparticles being attached to the SiN membrane. Upon imaging at higher magnifications the nanoparticles start moving along the SiN membrane and start to form agglomerates. Particle tracking was applied to 4 selected Au nanoparticles to study their movement.

Figures:

Complete “Plug & Play” Package. It includes (1) Nano-Cells, (2) Holder, (3) Stand, (4) Leak Tester (for full protection of TEM), (5) Syringe Pump and (6) Fluidic Connectors.

Nano-Cell. (a) The Nano-Cell consists of two chips which are sandwiched together to formed a sealed microfluidic compartment. (b) The dual-cell chip technology enables to locally load the sample directly on the field of view, as well as doing cell culture.

Functionalized Sample Holder. The tip has been designed to ensure the self-alignment of the chips. Furthermore, the tip closure is force-independent. Finally, the holder has a modular design which allows the user to detach i.e. the tip and replace the inner tubing.

Experimental Results. The Ocean System was used to study the Au nanoparticle agglomeration mechanisms in liquid. Upon imaging at higher magnifications the Au nanoparticle start moving along the SiN membrane and start to form agglomerates. Particle tracking was applied to 4 selected Au nanoparticles to study their movement.

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EMC Abstracts - https://emc-proceedings.com/abstract/the-ocean-system-microfluidic-based-system-for-in-situ-analysis-of-liquid-processes-inside-the-tem/