Controlled microaspiration for high‐pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

Abstract

SummaryHigh-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of highpressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.

Deep nuclear invaginations linked to cytoskeletal filaments: Integrated bioimaging of epithelial cells in 3D culture

“…4B). Note that owing to the specific sample preparation method chosen for the 2D TEM imaging, internal membranes showed low contrast (Giddings, 2003;McDonald and Morphew, 1993;Triffo et al, 2008). Also, owing to the difference in heavy metal staining needed to image samples with FIB-SEM, the texture of the chromatin within the nucleus appeared to be less heterogeneous in comparison to that upon staining for TEM (Fig.…”

“…2). The cellular ultrastructure showed exquisite preservation owing to the ultra-rapid freezing and the freeze-substitution process (McDonald and Auer, 2006;Triffo et al, 2008), otherwise known as high-pressure freezing with freeze substitution (HPF-FS). The compact spacing of elements within the cytoplasm ( Fig.…”

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

“…However, fresh tissue microdissection and the exposure of unfixed tissues to relatively high levels of cryoprotectants (e.g. 20% glycerol), just prior to high-pressure freezing, can lead to physical damage or osmotic stress (Triffo et al, 2008). To avoid aggregation artifacts caused by the dehydration step in conventional protocols, yet guard against osmotic stress in fresh dissected tissue sample upon exposure to cryoprotectants, we adopted a conservative approach to sample preparation.…”

“…Using the Leica automated freeze-substitution system AFS (Leica Microsystems, Vienna, Austria), cryofixed specimens were freeze-substituted in anhydrous acetone containing 1% osmium tetroxide and 0.1% uranyl acetate and after several rinses in pure acetone infiltrated with Epon-Araldite following established protocols (McDonald and Müller-Reichert, 2002;McDonald, 2007;Triffo et al, 2008). Specimens were flat-embedded between two microscopy slides and polymerized at 60˚C over 1 to 2 days.…”

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

“…Sosinsky et al [87] showed that chemical pre-fixation before HPF and FS followed by resin embedding provided a better preservation of ultrastructure than a single chemical fixation. However, exposure of fresh, microdissected tissue to highly concentrated cryo-protectants can lead to osmotic stress and physical damage [126]. Using aldehyde fixation before HPF-FS and resin embedding, Ewald et al [127] achieved high-quality ultrastructural preservation of cell membranes in multilayered epithelium.…”