Freeze‐substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

Abstract

SummaryHigh-pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high-pressure frozen, freeze-substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around -60• C.

Apoptotic-likeLeishmaniaexploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

“…Samples were freeze substituted in acetone containing 0.1% (w/v) uranyl acetate, 0.2% (w/v) osmium tetroxide and 5% (v/v) water and embedded in epon as described previously. 63,64 The samples were imaged with a Zeiss EM109 transmission electron microscope (Carl Zeiss, Oberkochen, Germany) at an acceleration voltage of 80 kV.…”

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

“…The problem becomes even more complicated, when the sample is planned to be used for subsequent protein detection by immunocytochemistry at the ultrastructural level, which also requires preservation of the antigen to be recognized by a specific antibody. A range of protocols are available for the freeze-substitution method and they have to be selected empirically since comparative studies are scarce and our mechanistic understanding of the substitution process barely exists (Buser and Walther 2008;Giddings 2003;Lonsdale et al 1999;Monaghan et al 2003;Takahashi et al 1997;Takizawa et al 1999;Wild et al 2001). These procedures are usually optimized to bring solution for a specific problem, and they vary in solvent polarity, substitution schedule, fixative additives, and resin embedment.…”

“…Freeze-substitution combines the benefits of the initial cryofixation with the ability to embed and section the samples for a variety of subsequent electron microscopy applications, morphological and immunocytochemical studies included (Giddings 2003;Monaghan et al 1998;Studer et al 2008). After cryofixation, freeze-substitution mainly influences the sample quality and it is probably the most difficult process to be exactly regulated (Buser and Walther 2008;Hawes et al 2007). This step deals with the classical dilemma that the sample has to be dehydrated without altering or displacing the biological structures.…”

Protein pUL128 of Human Cytomegalovirus Is Necessary for Monocyte Infection and Blocking of Migration

“…High-pressure freezing was performed with an HPF 01 freezing apparatus (Engineering Office M. Wohlwend GmbH, Switzerland). Samples were freeze substituted in acetone containing 0.1% (wt/vol) uranyl acetate, 0.2% (wt/vol) osmium tetroxide, and 5% (vol/vol) water and embedded in Epon (8,37). After being thin sectioned, samples were imaged with a Zeiss EM10 transmission electron microscope at an acceleration voltage of 80 kV.…”