Cryo-Planing Vs Freeze Fracture: Sample Preparation for Cryo-HRSEM

Irene Y T Chang, Derk Joester

Research output: Chapter in Book/Report/Conference proceedingConference contribution


Cryo-SEM is particularly efficient at revealing the ultrastructure of biological systems in a near-tonative
state and at nanometer resolution. Freeze fracture is conveniently used to prepare interior surfaces
of frozen-hydrated samples, but the random nature of fracture does not ensure the passage of the fracture
plane through the regions of interest [1, 2]. The resultant surface is rough, which can make interpreting
delicate structural features in the sample difficult. To address these issues, we developed the cryo triple
ion gun milling (CryoTIGM) technique. High pressure-frozen samples were trimmed using a custombuilt
cryo-saw to expose a sample edge, which was brought into contact with a milling mask. Three
broad Ar+ beams were aimed at the sample edge, and removed materials above the mask to create a
cross-section in the sample at the level of the mask. In this manner, large areas in frozen-hydrated
samples were cryo-planed in only a few hours. Cryo-planed samples were subsequently freeze-etched
and coated with Pt to increase contrast.

We evaluated sample preparation by CryoTIGM against freeze fracture for three biological systems,
yeast cell suspensions, mouse liver biopsies, and suspensions of whole sea urchin embryos. The fracture
plane frequently occurs between the leaflets of lipid bilayer membranes in freeze-fractured samples,
such as the plasma membrane and the nuclear membrane. While this process reveals intra-membranous
structures (e.g. nuclear pore complexes), structures inside organelles, for instance the cristae of the inner
membrane of mitochondria, cannot be accessed reliably (Fig. 1A, C). In contrast, surfaces cryo-planed
using CryoTIGM are very smooth and membranous compartments are always revealed in cross section
(Fig. 1B, D). Moreover, morphological information of membranous structures in the out-of-plane
direction becomes available. For example, nuclear pores appear as discontinuities in the nuclear
membrane in ion-milled samples (Fig. 1D).

Freeze fractured sample surfaces are expectedly rough and provide topographical information about the
3D arrangement. However, this at times complicates interpretation. This shortcoming is exemplified in a
comparison of freeze-fractured and CryoTIGM-prepared sea urchin embryo samples (Fig. 2). While both
techniques show the ectoderm, the enveloping hyaline layer, and the overall packing of ectodermal cells
(Fig. 2A, B), tight contacts among neighboring ectodermal cells, suggesting the presence of intercellular
junctions, can be identified only after CryoTIGM (Fig. 2B) Membrane tethers extended from ectodermal
cells to the hyaline layer are now visible. The hyaline layer is resolved to consist of two layers, with the
space between them occupied by some kind of vesicles or granules. In turns of image resolution and
contrast, however, we observe both techniques capable of providing cellular and organellar details at
high resolutions and with good contrast. Fundamental components, such as the nucleus, mitochondria,
the endoplasmic reticulum, and the Golgi apparatus, can be visualized via both methods.

In conclusion, cryo-planing of frozen-hydrated samples by CryoTIGM is a convenient way to prepare
very large, smooth surfaces for subsequent analysis by cryo-HRSEM (and possibly other cryogenic
imaging techniques). The ultrastructure of cells and tissues is well preserved and can be imaged at high
resolution and with good contrast. Given the large area and high quality of the surfaces, the relatively
fast sample preparation, and the excellent preservation of ultrastructural features, CryoTIGM is a valuable addition to the toolbox that includes freeze-fracture, cryo-FIB-SEM, and cryo-EM.
Original languageEnglish (US)
Title of host publicationProceedings of Microscopy & Microanalysis 2015
Number of pages2
StatePublished - 2015


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