Microfluidic Liquid Cell with Silicon Nitride Super-Thin Membrane for Electron Microscopy of Samples in Liquid

• Published in: Biosensors (Basel) Vol. 12; no. 12; p. 1138
• Main Authors: Sugihara, Akihiko, Ishida, Tadashi
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.12.2022; MDPI
• Subjects: Analysis; Atmospheric pressure; Bacteria; Bacterial infections; Biological properties; Biological samples; Care and treatment; Communicable diseases; Composition; Coronaviruses; COVID-19; Cyanobacteria; Diagnosis; Dimethylpolysiloxane; Disease transmission; Drug resistance; Electron beams; Electron microscopy; Etchants; Etching; Experiments; High resolution; in-liquid scanning electron microscopy; liquid cell; Membranes; Microchannels; Microfluidics; Microfluidics - methods; Microscopy, Electron; Monte Carlo simulation; PDMS microchannel; Phages; Polydimethylsiloxane; Properties; Scanning electron microscopy; Scattering; Silicon; Silicon compounds; Silicon Compounds - chemistry; Silicon nitride; Silicon substrates; super-thin electron transparent membrane; virus/bacteria; Visualization; Japan; liquid cell; PDMS microchannel; virus/bacteria; in-liquid scanning electron microscopy; super-thin electron transparent membrane
• ISSN: 2079-6374; 2079-6374
• DOI: 10.3390/bios12121138

Microfluidic liquid cells have been developed to visualize nanoscaled biological samples in liquid using a scanning electron microscope (SEM) through an electron-transparent membrane (ETM). However, despite the combination of the high-resolution visualization of SEM and the high experimental capability of microfluidics, the image is unclear because of the scattering of the electron beam in the ETM. Thus, this study developed a microfluidic liquid cell with a super-thin ETM of thickness 10 nm. Because the super-thin ETM is excessively fragile, the bonding of a silicon-nitride-deposited substrate and a polydimethylsiloxane microchannel before silicon anisotropic etching was proposed prevented the super-thin ETM from damage and breakage due to etching. With this protection against etchant using the microchannel, the yield of the fabricated super-thin ETM increased from 0 to 87%. Further, the scattering of the electron beam was suppressed using a microfluidic liquid cell with a super-thin ETM, resulting in high-resolution visualization. In addition, T4 bacteriophages were visualized using a super-thin ETM in vacuum. Furthermore, the cyanobacterium sp. PCC6803 in liquid was visualized using a super-thin ETM, and sub-microscopic structures on the surface were observed.