Self-aligned wet-cell for hydrated microbiology observation in TEM

Area of Science:

• Materials Science
• Microscopy
• Biotechnology

Background:

• Transmission electron microscopy (TEM) traditionally requires samples to be in a vacuum, limiting the observation of dynamic biological processes.
• Existing wet cell technologies face challenges with sample alignment, stability, and electron beam-induced artifacts.

Purpose of the Study:

• To develop and demonstrate a novel Self-Aligned Wet (SAW) cell for in-situ, real-time observation of biological samples in a liquid environment within a TEM.
• To overcome limitations of existing wet cells, including alignment difficulties and electron beam interactions with the sample chamber.

Main Methods:

• Fabrication of the SAW cell using bulk-micromachining, creating two complementary frames (in-frame and out-frame) with silicon nitride observation windows.
• Utilizing surface tension of a bio-sample droplet for self-alignment of the in- and out-frames, eliminating the need for external positioning stages.
• Designing thin silicon nitride membranes to withstand pressure differences and allow electron penetration, incorporating a novel approach to mitigate charging/heating issues.

Main Results:

• Successful demonstration of a self-aligned liquid chamber enclosed by silicon nitride membranes within a TEM.
• Achieved a large field of view (150 μm × 150 μm) enabled by a unique circular membrane formation process.
• Presented TEM observations of Deinococcus radiodurans growth in a 20 μm thick liquid chamber within the SAW cell, showcasing its capability for dynamic biological studies.

Conclusions:

• The Self-Aligned Wet (SAW) cell provides a robust and user-friendly platform for high-resolution in-situ TEM imaging of biological samples in liquid.
• The self-alignment mechanism and novel design address key challenges, paving the way for advanced studies of cellular processes in their native wet environments.
• The SAW cell is suitable for observing microbial growth and other dynamic biological phenomena under realistic conditions within a TEM.