Abstract text (incl. figure legends and references)

We introduce the "Stream System" to enable in-situ liquid phase transmission electron microscopy (LPTEM). The Stream system raises the impact in single-particle research as well as in pharmaceutical development, as it overcomes historical limitations and enables real-time studies of biological processes, in their native liquid state, as a function of different stimuli.

The "Stream System" is a plug-and-play system for TEMs. It relies on a customized side-entry holder with integrated tubing to supply liquid into a microfluidic Lab-on-a-Chip device acting as a smart specimen carrier, referred to as the Nano-Cell, which contains on-chip inlet and outlet to create a defined fluidic path, as well as an integrated set of biasing electrodes or a microheater. The liquid control is enabled via a Liquid Supply System (LSS), which can independently control the pressure at the inlet and the outlet. The fluid containing the sample (e.g. liposome, vesicles, viruses, proteins, etc) is flown from the LSS into the inlet of the sample carrier to initialize the in-situ experiment. The fluid coming out of the outlet is then re-collected in an empty Eppendorf tube on the LSS, to enable downstream biochemical analysis. These capabilities enable dynamic studies for life science and pharmaceutical research as a function of temperature/biasing/pressure/flow rate.

The system enables high level of control over the microfluidics and high throughput studies. In the case of Single Particle Analysis (e.g. SPA of proteins), the Brownian motion of the particle can be used to resolve its 3D structure while being in liquid. This technology has therefore enabled the possibility to resolve the 3D structure of apoferritin and an RNA polymerase while being in liquid. Furthermore, the user can avoid this liquid-air interface. Additionally, the system allows to control the mass transport, enabling the user to optimize the biochemistry on-the-fly. Liquid phase TEM can be used to visualize in-situ the synthesis of liposomes (e.g. for drug delivery purposes). The tip of the liquid holder is removable, creating a "Lab-on-a-Cartridge" (i.e. tip + Nano-Cell) that can be independently used to enable interoperability with other tools (e.g. SEM, Fluorescent Microscope, Synchrotron, etc.) and correlative studies.

Our development provides the unique possibility to visualize biological processes in real time with high resolution, while the sample is in its native environment (as a function of different stimuli). This opens up several possibilities such as SPA (proteins, viruses, exosomes, etc), biomedical/pharmaceutical applications (e.g. disease biomarkers, drug delivery vehicles or vaccines) and fundamental studies in biophysics and biochemistry. We believe that this technique will become a very powerful complementary technique to cryo electron microscopy.

References:

[1] G Battaglia et al, "4D imaging of soft matter in liquid water" (2021), DOI:10.1101/2021.01.21.427613

[2] J Cookman, "Visualising early-stage liquid phase organic crystal growth via liquid cell electron microscopy" Nanoscale, 7 (2020).

[3] A Ianiro, et al. "Liquid–liquid phase separation during amphiphilic self-assembly", Nature Chemistry, 11, 320–328 (2019), https://doi.org/10.1038/s41557-019-0210-4

[4] A Rizvi, "Observation of Liquid–Liquid-Phase Separation and Vesicle Spreading during Supported Bilayer Formation via Liquid-Phase Transmission Electron Microscopy", Nano Letters, 10325–10332 (2021)