Liquid-phase 3D electron microscopy for materials science and biology – Liquid3DSTEM

The project involves first of all the development of a theoretical model to predict the spatial resolution as a function of the experimental parameters, as well as a method of volume reconstruction adapted to the series of images acquired rapidly and at low dose (WP1). A second work program (WP2) is focused on experimental developments, in environmental scanning microscopy (ESEM) and environmental transmission microscopy (ETEM). Finally, two parallel studies will be conducted in liquid 3D STEM to demonstrate and promote its unique capabilities: one in materials science on latex and surfactant suspensions (WP3), and the other in biophysics to study the fate of nanoparticles absorbed by biological cells (WP4).

The first half of the project was devoted on the one hand to the development of a theoretical model allowing the computation of the spatial resolution according to the experimental parameters used during the experiments (WP1). On the other hand, a large experimental development has been provided to allow the adjustment and automatic acquisition of series of images in ESEM, via a code in Python (WP2). In parallel, the Python code is being adapted for the acquisition of image series in ETEM (WP2). An improvement of the ESEM stage has also been made to improve the alignment of the detector, which was not initially planned. The ESEM system is now operational. Different samples from material science and biology have been collected and are being studied with the different systems available (ESEM, ETEM, liquid cell) (WP3 and WP4).

The main feature at this stage is the development of the Python code. This code allows monitoring both the microscope and the home-made sample holder. Thanks to automation, it is possible to quantify the electronic dose received by the sample. Moreover, the acquisition time is considerably reduced (to about 3 minutes for a hundred images instead of about 1 hour for a dozen images), which also reduces the received dose and should allow the study of sensitive samples in 3D.
In the second part of the project, the study of materials of different natures will allow a refinement and the validation of the method, as well as an objective comparison of the different 3D liquid STEM techniques. The results thus obtained will be presented in publications and in scientific conferences.
The experimental developments carried out within the project have already enabled to meet the needs expressed by laboratories and companies (call for experiments via the national METSA network), whether for the study of liquid samples or for 3D studies on dry samples.

- 1 mono-partner paper submitted in 2022
- 1 communication in an international conference with publication of the abstract in a peer-reviewed journal (CISCEM, Paris, 2021)
- 2 communications in international conferences (MC2021 online, 16MCM in Czech republic in septembeer 2022)
- 3 communications in national conferences

Electron microscopy has traditionally been associated with the study of thin, solid samples in vacuum. In the last decade, a few groups pioneered systems achieving nanometer resolution for liquid specimens. The broad applicability of liquid phase electron microscopy (LPEM) has driven a wave of interest as it has opened exciting possibilities for solving grand challenges in materials science,chemistry, biology, and other fields, and has opened the route for operando studies. Whatever the application field, a three-dimensional (3D) representation of the sample in liquid is often required for understanding its structure. The primary method currently used for obtaining insight into the 3D structure at the nanometer scale of unique samples from biology and materials science is tilt-series transmission electron microscopy. In particular, scanning transmissionelectron microscopy (STEM) is well-adapted to the study of thick specimens such as liquid samples. The goal of this proposal is to establish Liquid 3D STEM as new microscopy modality, presenting anunique way for nanoscale characterization of samples in liquid from both materials science and biology. Liquid 3D STEM has enhanced capabilities compared to state-of-the-art 3D electron microscopy because it provides a means to study specimens in liquid. The projectbenefits from the synergy of two leading centers for in situ electron microscopy, INSA in Lyon, with a focus on materials science, and INMin Saarbrücken (Germany), with research focused towards biophysics. The technique will be jointly developed by gathering the complementary expertise of both groups. Each group will use Liquid 3D STEM to study a system with which it has already several years ofexperience. By combining efforts and expertise, we expect to be able to overcome the main difficulties encountered during the experiments (sensitivity to the electron beam, low contrast, large thickness).