Freeze fracture direct imaging: A new freeze fracture method for specimen preparation in cryo-transmission electron microscopy

In this paper, we present a new freeze fracture method for specimen preparation for transmission electron microscopy frozen samples. We call it freeze fracture direct imaging (FFDI) because it is a hybrid of conventional freeze fracture electron microscopy (FFEM) and cryo-transmission electron microscopy (cryo-TEM), combining elements of the fracture technique with direct imaging. Like in FFEM, the sandwich method is used to prepare the sample in a protected fashion. However, after the sample is vitrified and fractured, it is not shadowed but directly imaged. The new technique avoids some experimental artifacts produced by the blotting procedure in conventional cryo-TEM. It relies, though, on occasional fractures transparent to the electrons. The advantageous features are demonstrated by a comparison between conventional cryo-TEM and FFDI micrographs of vesicular solutions. The second outstanding advantage over conventional cryo-TEM is the fact that it is now possible for the very first time to directly image oil-rich mixtures films which normally would dissolve in the cryo-medium ethane. Micrographs of pure oil and of oil-rich microemulsions clearly prove the reliability of the FFDI technique as well as its enormous potential.