Reproducible Bare Grid Technique

New Plunge Freezer for Cryo-TEM

Ian Lamswood, Leica Microsystems

Imaging neurons, macromolecular assemblies, or viruses in their native, hydrated environment in the cryo-transmission electron microscope (cryo-TEM), is the state-of-the-art technique in electron microscopy. It provides maximum resolution with minimal specimen damage. The plunge freezer Leica EM GP helps to standardise procedures and to improve reproducibility.

Many specimens for cryo-TEM can be prepared by immersion freezing, where a fluid sample is pipetted onto an EM grid (usually coated), the excess removed until a thin film remains before plunging into a cryogen such as liquid ethane. The grid can then be directly transferred under cryo conditions to the cryo-TEM for observation.

This simple technique has been modified over time, creating a procedure carried out by many with homemade devices in an attempt to standardise the procedure. Although a simple method, it is imperative that the film thickness on the grid can be reproduced, vitreous ice can be formed and low contamination observed; otherwise much time is wasted loading useless samples into the cryo-TEM. The sample film is only tens to hundreds of nanometres thick and so is affected by temperature shifts and humidity. If the humidity is too low then the film breaks due to it quickly drying. If the sample is adversely affected by temperature then the morphology may change before freezing.

Easy freezing

Leica Microsystems has developed a plunge freezer in conjunction with Dr. Günter Resch of the IMP/IMBA Electron Microscopy Facility in Vienna, Austria, to standardise procedures and make the bare grid technique more reproducible. The Leica EM GP plunge freezes samples into liquid ethane after removing excess fluid by automatic blotting. Prior to freezing the sample is maintained in a temperature and humidity controlled protective environmental chamber.

Access ports on both sides of the environmental chamber allow easy pipetting of solutions and suspensions for both left and right handed users. The excess fluid is then removed by automatic blotting with filter paper from one side of the grid. Single sided blotting was developed to prevent damage to delicate support films. The blotting and plunging can be initiated via touch screen or footswitch. The complete procedure can be viewed via a stereomicroscope for full control of all processes with LED illumination into the chamber and down to the Dewar enhancing observation.

Safety first

After freezing the grid is transferred to a pre-cooled grid box inside a transfer container filled with liquid nitrogen (LN2) which can be taken to the specimen holder of the cryo-TEM to prepare for loading into the electron microscope. Operation is via touch screen control with all adjustable parameters visible. A programme library is available with storage capabilities for working parameters including blotting time and delay time before freezing. The Leica EM GP operates under strict safety conditions. An alarm signals when either the secondary cryogen is too warm or the LN2 level is too low. At the end of a run the instrument has a bake-out cycle for both the environmental chamber and Dewar to dry all components.

Highly flexible

The Leica EM GP has been made for all EM laboratories with a need to view vitrified fluid samples or extremely thin samples in the cryo-TEM, including biological research, virology, protein crystallography, pharmaceutical labs, cosmetic companies and industrial labs to prepare for example suspensions such as in paint research or solutions and emulsions in both aqueous and inorganic solvents. It can be used to plunge freeze samples on EM grids, sapphire discs and samples in freeze fracture planchettes but the main application is bare grid technique.