An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy.

Visualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

Microstructural observation of the swollen catalyst layers of fuel cells by cryo-TEM.

It is important to understand and control the fine structure of the fuel cell catalyst layer in order to improve the battery characteristics of the fuel cell. A major challenge in observing the microstructure of the catalyst layer by electron microscopy is the visualization of ionomers, which have low contrast and are susceptible to damage by electron beam irradiation. Previous papers have reported transmission electron microscopy (TEM) observations of ionomers neutralized with cesium (Cs) ions. However, this approach involves chemical reactions and indirect visualization of ionomers. In contrast, we have previously revealed the microstructure of ionomers in frozen catalyst inks by cryogenic (cryo) scanning electron microscopy and cryo-TEM. In general, ionomers are basically used under high-temperature and humid conditions while the fuel cell is operating. Therefore, in this study, ultrathin sections prepared from the fuel cell catalyst layer (membrane electrode assemblies) were incubated in a chamber under high-temperature and humid conditions and then rapidly frozen for observation by cryo-TEM. As a result, we succeeded in observing the pore structure of the catalyst layer in the swollen state of the ionomer. The swollen ionomer surrounded and enclosed the Pt/C aggregates and bridged over the pores in the catalyst layer.

A simple flash and freeze system for cryogenic time-resolved electron microscopy.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

Microstructural observation of fuel cell catalyst inks by Cryo-SEM and Cryo-TEM.

In order to improve the electricity generation performance of fuel cell electric vehicles, it is necessary to optimize the microstructure of the catalyst layer of a polymer electrolyte fuel cell. The catalyst layer is formed by a wet coating process using catalyst inks. Therefore, it is very important to observe the microstructure of the catalyst ink. In this study, the morphology of carbon-supported platinum (Pt/C) particles in catalyst inks with a different solvent composition was investigated by cryogenic scanning electron microscopy (cryo-SEM). In addition, the morphology of the ionomer, which presumably influences the formation of agglomerated Pt/C particles in a catalyst ink, was investigated by cryogenic transmission electron microscopy (cryo-TEM). The results of a cryo-SEM observation revealed that the agglomerated Pt/C particles tended to become coarser with a higher 1-propanol (NPA) weight fraction. The results of a cryo-TEM observation indicated that the actual ionomer dispersion in a catalyst ink formed a network structure different from that of the ionomer in the solvent.