Advanced Phase Plate Fabrication and Thin Film Characterization for TEM Imaging of Biological Samples

Phase plates (PPs) for transmission electron microscopy (TEM) have been a research field since 2001 [1] and are of continuing interest to enhance contrast and increase resolution when imaging biological samples [2]. The most promising PPs for soft-matter imaging are thin-film-based [3]. These include Volta-, Zernike-, and Hilbert-type PPs. Thin-film PPs are generally fabricated by hand using sputtering tools in a single-device process [4]. These PPs suffer from several limitations that plague their widespread implementation. These challenges include contamination, ease of installation and use, lack of reproducibility, and lack of full understanding and control of the induced phase shift. In this work we are using cleanroom processes to fabricate PPs that are reproducible with known parameters. In this manner we can both know and control the induced phase shift. We present the fabrication of both Zernike-type phase plate devices (ZPPs), with an ideal phase shift of <i>Δφ = π/2</i>, and Hilbert-type PP devices (HPPs), for which <i>Δφ = π</i>.<br/><br/>In order to achieve this, it is necessary to know the accurate mean inner potential (MIP) of the materials involved. For our work, the most promising thin film materials are silicon nitride and amorphous carbon. MIP values for these materials in the literature vary widely, making it difficult to fabricate a device with the ideal phase shift. The phase shift is related to the mean inner potential <i>V₀</i> and the film thickness <i>t</i> (via an electron constant <i>C_E</i>) in the following way [5]: <i>Δφ = C_EV₀t</i>. We use ellipsometry and AFM techniques to determine and verify the thicknesses of the thin films. We use electron holography (EH) [6] to measure the phase shift of our devices. Using the thickness and phase shift values we can then accurately determine the MIP of our thin films. We can further use this to optimize our devices for optimal phase shift.<br/><br/>In addition to our material characterization, we installed the PPs in the back-focal plane of a <i>TF Tecnai TEM</i> using the existing objective aperture mechanism. We will demonstrate the functionality of our phase plate devices via contrast enhancement, using both a test sample made from thin carbon structures on a standard carbon-film TEM grid, and biological samples at cryogenic temperatures.<br/><br/><u>REFERENCES </u><br/>[1] R. Danev, K. Nagayama, Transmission electron microscopy with Zernike phase plate, 2001, Ultramicroscopy 88: 243<br/>[2] M. Obermair et al., Analyzing contrast in cryo-transmission electron microscopy: Comparison of electrostatic Zach phase plates and hole-free phase plates, 2020, Ultramicroscopy 218: 113086<br/>[3] R. M. Glaeser, Methods for imaging weak-phase objects in electron microscopy, 2013, Rev. Sci. Instrum. 84: 111101<br/>[4] R. Danev et al., Practical factors affecting the performance of a thin-film phase plate for transmission electron microscopy, 2009, Ultramicroscopy 109: 312<br/>[5] M. Malac et al., Phase plates in the transmission electron microscope: operating principles and applications, 2021, Microscopy Vol. 70, No. 1: 75<br/>[6] M. N. Yesibolati et al., Mean Inner Potential of Liquid Water, 2020, Phys. Rev. Lett. 124: 065502