Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

We present a process for batch fabrication of a novel scanning microscopy probe for thermal and magnetic imaging using standard micromachining and conventional optical contact lithography. The probe features an AFMtype cantilever with a sharp pyramidal tip composed of four freestanding silicon nitride nanowires coated by conductive material. The nanowires form an electrical cross junction at the apex of the tip, addressable through the electrodes integrated on the cantilever. The cross junction on the tip apex can be utilized to produce heat and detect local temperature changes or to serve as a miniaturized Hall magnetometer enabling, in principle, thermal and magnetic imaging by scanning the probe tip over a surface. We have successfully fabricated a first probe prototype with a nanowire tip composed of 140 nm thick and 11 µm long silicon nitride wires metallized by 6 nm titan and 30 nm gold layers. We have experimentally characterized electrical and thermal properties of the probe demonstrating its proper functioning.978-1-4244-5764-9/10/$26.00 ©2010 IEEE

No abstract

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

No abstract