Inner ear tissue preservation by rapid freezing: Improving fixation by high-pressure freezing and hybrid methods

Abstract

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues.

High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle

“…Follicles make an unusual challenge for HPF because, at approximately 200 µm diameter, they are close to the maximum dimensions possible with HPF. Although HPF is known to achieve tissue preservation to greater depths than other rapid-freeze fixation methods, and instrument manufacturers sometimes claim that 200 µm thick samples can be routinely processed, the success of the method is sample dependent and 50 µm preservation of thick samples is a more realistic expectation (Studer et al, 2001;Studer et al, 2008;Bullen et al, 2014). However, follicles are likely to have two advantages over similarly thick blocks of tissue.…”

“…For example, we are concerned that diffusion of both paraformaldehyde and glutaraldehyde (and additional reagents such as osmium, tannic acid and wash buffers) into follicles, and diffusion of water and dehydrating solvents out of follicles may be uneven, resulting in distortions to the fine follicle structure making it inadequate for ultrastructural analysis. HPF in combination with FS (HPF-FS) has shown to be very effective in preserving the ultrastructure of cochlea, a large and structurally complex tissue (Bullen et al, 2014). Here we investigated the feasibility of replacing chemical conventional methods with HPF and dehydration with FS for preserving the ultrastructure of developing wool fibres.…”

“…In addition, we also investigated the effects of prefixation on follicles. Prefixation of tissues with aldehydes prior to HPF has been shown to benefit the preservation of some highly labile and delicate structures, such as stereocilia and actin fibres (Meissner & Schwarz, 1990;Sosinsky et al, 2005Sosinsky et al, , 2008Bullen et al, 2014). The formation of keratin in follicles occurs as various fine networks and aggregates of keratin intermediate filaments (KIFs) that differ in their internal arrangements, making prefixation a potentially useful step.…”

Improving Biomaterials Imaging for Nanotechnology: Rapid Methods for Protein Localization at Ultrastructural Level

“…Although some specific contributions have displayed a time‐, material‐saving, and improved results; the optimization of TEM methods is clearly not enough achieved, especially for biomaterials. In this context, pre‐ and post‐embedding immunolabeling protocols for TEM are a key question to link biochemistry, molecular biology, structural, and ultrastructural studies or other newer research techniques with the spatio‐temporal localization of peptides and proteins in a quantitative manner at nanoscale . Here, we have developed two new post‐embedding iEM methods for peptide and protein biomaterials based on the most used conventional approach for TEM immunolabeling, aimed to the reduction of time, reagents, and steps of labeling, that would in turn reduce costs, residues, and artifacts and increase protein localization (then, method efficiency and efficacy) of less altered nanobiomaterials.…”

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy

“…Images of the complex architecture of stereocilia bundles at the resolution of single nanometers can be obtained using scanning or transmission electron microscopy (SEM or TEM, correspondingly) ( 8-10 ) . However, the samples for electron microscopy are “dead”, since they typically undergo chemical fixation and dehydration processes.…”