In Situ Transmission Electron Microscopy of Light-Induced Processes in Liquid

<i>In situ</i> transmission electron microscopy (TEM) allows real-time observation of dynamic processes. Although stimuli such as strain, temperature, and magnetic or electric fields are commonly explored, the interaction between light and matter is less frequently studied. Nevertheless, recent years have witnessed a notable gain in interest in this aspect of the technique. Historically, the delivery of light into TEM samples has been a complex task, with various methods such as dedicated specimen holders, parabolic mirror mounts, or optical fibers being employed. However, these solutions often have limitations, such as uneven sample illumination, restrictions on sample manipulation, or issues with light intensity calibration [1].<br/><br/>Due to the limitations of commercial solutions, we created our own sample illumination in our TEM systems. Our first setups used the Hitachi H-800 [2], and allowed for precise control over light exposure and dose measurement [3]. They allowed us to perform a series of preliminary studies and define precise requirements for the final configuration of the TEM microscope for light-induced studies.<br/><br/>Our main trials focused on imaging the antimicrobial photodynamic therapy (aPDT) phenomena [4]. aPDT is a modern, noninvasive method for combating infections, including those caused by drug-resistant bacteria. The process relies on photosensitizers (PS) that, when exposed to specific light wavelengths, generate reactive oxygen species (ROS), which damage cellular structures. The primary targets of ROS are cell membranes, leading to functional disorders and bacterial inactivation. However, damage to nucleic acids and proteins can also play a role in this process. To enhance the understanding of the mechanism of therapy, advanced techniques such as electron microscopy are necessary, especially since light microscopy often lacks the resolution required to observe the fine details of microbial damage. For this purpose, we also had to implement liquid cell TEM preparation, based on the SiN + graphene configuration and on sandwitches of amorphous carbon (aC) films. In a study designed to explore the aPDT, bacteria were encapsulated with liquid photosensitizers and irradiated with light in a TEM setup. The system used in the experiments included a top-mounted light source, allowing precise control over illumination. For these tests, Gram-positive Staphylococcus aureus and Gram-negative Acinetobacter baumannii were used, with methylene blue as photosensitizer and 660nm light illumination. One of the major issues in using TEM to observe hydrated samples, such as bacteria in a liquid cell, is the damage caused by the electron beam itself. In preliminary tests, the team successfully demonstrated that light-induced effects could be isolated from electron-induced damage. The results showed that aPDT damage occurred mainly in the outer cellular structures of the bacteria, confirming the central role of membrane disruption in aPDT.<br/><br/>In cooperation with ThermoFisher Scientific, we managed to create a new model of the illuminator on the Talos F200i microscope. The ongoing delivery of the chip-based liquid cell holder will allow us to compare the imaging efficiency of light- and electron-beam-induced processes in different types of liquid cells (SiN/graphene/aC). The newly implemented project will answer the questions of how different photosensitizers act on microorganisms of the ESKAPE group.<br/><br/>This research was made possible by the National Science Center, Poland (2023/51/D/ST11/01490). Andrzej Zak would like to acknowledge the Polish-American Fulbright Commission and Institute of International Education (Fulbright Senior Award) and LightTEM project (7308/IA/SP/2022, Ministry of Education, Poland).<br/><br/>[1] Zak A., Nano Letters, DOI: 10.1021/acs.nanolett.2c03669<br/>[2] Zak A. et al., Ultramicroscopy, DOI: 10.1016/j.ultramic.2021.11338<br/>[3] Zak A., Micron, DOI: 10.1016/j.micron.2021.103058<br/>[4] Muehler D. et al., DOI: 10.3389/fmicb.2020.589364