Improved Microchip Design and Application for In Situ Transmission Electron Microscopy of Macromolecules

Abstract

Understanding the fundamental properties of macromolecules has enhanced the development of emerging technologies used to improve biomedical research. Currently, there is a critical need for innovative platforms that can illuminate the function of biomedical reagents in a native environment. To address this need, we have developed an in situ approach to visualize the dynamic behavior of biomedically relevant macromolecules at the nanoscale. Newly designed silicon nitride devices containing integrated "microwells" were used to enclose active macromolecular specimens in liquid for transmission electron microscopy imaging purposes.We were able to successfully examine novel magnetic resonance imaging contrast reagents, micelle suspensions, liposome carrier vehicles, and transcribing viral assemblies. With each specimen tested, the integrated microwells adequately maintained macromolecules in discrete local environments while enabling thin liquid layers to be produced.

Opportunities and challenges in liquid cell electron microscopy

“…In each design, the electrode materials and geometry can be customized for the particular applications (14,16,18,(20)(21)(22). A heating element (23) or cooling capability (24) can be integrated, and the silicon nitride surfaces can be patterned or chemically modified to enable reactions with species in solution (25)(26)(27)(28). Careful assembly procedures and cleaning are necessary (29).…”

“…One approach is to closely encase the material with graphene, minimizing the membrane thickness and the volume of surrounding liquid (105,106). Another approach is to use patterned silicon nitride substrates with microwells that control the liquid thickness (25,26) and additionally to tether biostructures by functionalizing the substrate (26)(27)(28).…”

Real-time observation of protein aggregates in pharmaceutical formulations using liquid cell electron microscopy

“…Under low-dose conditions (<1 electron per Å 2 ) specimens containing fragile RNA strands remained intact while in mild buffer solution. 11,18 Thus, local variances due to radiolysis can be somewhat attenuated by the use of solution that resists changes in pH, as recently demonstrated in theory and in practice. 19 …”

Through a Window, Brightly: A Review of Selected Nanofabricated Thin-Film Platforms for Spectroscopy, Imaging, and Detection

“…200 The available fabrication tools for Si-based cells, coupled with favorable material properties, allow for other functional architectures such as integrated microwells instead of a single sample volume. 201 Beyond structural modifications of the basic nanofluidic platform, advanced detection schemes can be used to compensate for some material and fabrication limitations, and implementation of different experimental techniques can broaden the achievable insights (Fig. 1a).…”