Environmental electron tomography is a sophisticated characterisation technique. Samples can be imaged in 3D at the nanometre scale using tilt-series acquisition, in their native liquid state or under partial gas pressure, thus simplifying sample preparation [1, 2, 3]. Combined with the STEM mode, it provides a wealth of structural and chemical information, while the deposited dose in STEM is known to be lower than in TEM mode [4].

Electron dose and acquisition time are the two main pending issues [5]. Firstly, the electron dose should be reduced and quantified, to preserve the sample architecture by reducing irradiation damage. Secondly, the acquisition time should be reduced in the same way and made proportional to the physical events to be considered, so that it becomes possible to monitor in 3D the changes in the sample during a chemical reaction or a physical process. Both issues have been addressed together and have led to a new acquisition protocol, called Liquid 3D-STEM.

The protocol is available for both Environmental Scanning and Transmission Electron Microscopies (ESEM and ETEM). It is based on the automated acquisition of long-range tilt series in a few minutes with a controlled electron dose and for a sample in its native liquid or environmental state. Simultaneous multi-mode acquisition is also possible (SE, BF, (HA)ADF).

We will present the keys steps of the Liquid 3D-STEM protocol, with results obtained on a ThermoFisher QuattroS ESEM and a ThermoFisher Titan ETEM. In both cases, the sample liquid state is preserved dynamically, by controlling the sample temperature and the environment pressure. We will show a custom software to run the automated tilt-series acquisition (M-SIS). The code is written in Python and uses the Autoscript (ESEM) and Digital Micrograph (ETEM) libraries. It provides automatic eucentric correction, dynamical drift correction and provides some other useful tools. Tilted image series post-processing and tomogram reconstructions are performed in ImageJ - with TomoJ - and 3D Slicer.

We will show results obtained with Liquid 3D-STEM on various types of beam-sensitive samples: aqueous SBA latex suspensions, various types of hydroxide wet gel suspensions and amphiphilic block copolymer nanorods. The 3D models of the samples show that it is possible to quantify sample structure in liquid with a spatial resolution of about 10 nm (ESEM) or about 1 nm (ETEM). Moreover, the electron dose can be kept below the degradation threshold of the samples. The limits of the protocol will be discussed in terms of liquid state stability, spatial resolution, sample thickness, sensitivity to electron dose and total experiment time.

References

[1] T. Schuh, N. de Jonge; Liquid scanning transmission electron microscopy: Nanoscale imaging in micrometers-thick liquids, Comptes Rendus Physique, 2014, 15(2-3).

[2] K. Masenelli-Varlot et al; Wet-STEM Tomography: Principles, Potentialities and Limitations, Microscopy and Microanalysis, 2014, 20(2).

[3] N. de Jonge; Theory of the spatial resolution of (scanning) transmission electron microscopy in liquid water or ice layers’, Ultramicroscopy, 2018, 187.

[4] S. Koneti et al; Fast electron tomography: Applications to beam sensitive samples and in situ TEM or operando environmental TEM studies. Materials Characterization 151, 2019, 480–495.