Nanofluidic and monolithic environmental cells for cryogenic microscopy

We present a device capable of combining nanofluidics and cryogenic transmission electron microscopy (cryo-TEM) to allow inspection of water-soluble samples under near-native conditions. The devices can be produced in a multitude of designs, but as a general rule, they consist of channels or chambers enclosed between two electron-transparent silicon nitride windows. With the appropriate design, those devices can allow screening of multiple samples in parallel and remove the interaction between the sample and the environment (no air-water interface). We demonstrate channel sizes from 80 to 500 nm in height and widths from 100 to 2000 μm. The presented fabrication flow allows producing hollow devices on a single wafer eliminating the need of aligning or bonding two half-cavities from separate wafers, which provides additional resistance to thermal stress. Taking advantage of a single-step through-membrane exposure with a 100 keV electron beam, we introduced arrays of thin (10-15 nm) electron-transparent silicon nitride membrane windows aligned between top and bottom (200-250 nm) carrier membranes. Importantly, the final devices are compatible with standard TEM holders. Furthermore, they are compatible with rapid freezing of samples, which is crucial for the formation of vitreous water, hence avoiding the formation of crystalline ice, that is detrimental for TEM imaging. To demonstrate the potential of this technology, we tested those devices in imaging experiments verifying their applicability for cryo-TEM applications and proved that vitreous water could be prepared through conventional plunge freezing of the chips.