Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

in Nematology
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At present, the importance of sample preparation equipment for electron microscopy represents the driving force behind major breakthroughs in microscopy and cell biology. In this paper we present an introduction to the most commonly used cryo-fixation techniques, with special attention paid towards high-pressure freezing followed by freeze substitution. Techniques associated with cryo-fixation, such as immunolocalisation, cryo-sectioning, and correlative light and electron microscopy, are also highlighted. For studies that do not require high resolution, high quality results, or the immediate arrest of certain processes, conventional methods will provide answers to many questions. For some applications, such as immunocytochemistry, three-dimensional reconstruction of serial sections or electron tomography, improved preservation of the ultrastructure is required. This review of nematode cryo-fixation highlights that cryo-fixation not only results in a superior preservation of fine structural details, but also underlines the fact that some observations based on results solely obtained through conventional fixation approaches were either incorrect, or otherwise had severe limitations. Although the use of cryo-fixation has hitherto been largely restricted to model organisms, the advantages of cryo-fixation are sufficiently self-evident that we must conclude that the cryo-fixation method is highly likely to become the standard for nematode fixation in the near future.

Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

in Nematology



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    Main transmission electron microscopy preparation pathways, with focus on cryo-fixation and related applications.

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    A-C: Examples of cryo-fixation (high pressure freezing followed by freeze substitution) vs chemical fixation (A′, B′, C′). A: longitudinal section of the cuticle of Acrobeles complexus; B: Detail of two abutting membranes of A. complexus sperm, the phospholipid bilayer is visible after cryo-fixation; C: Immature spermatozoa in testis of Trichodorus similis (am = amorphous, fb = fibrous bodies, n = nucleus). D, E: Examples of typical cryo-fixation applications; D: Black dots show immunolocalisation of major sperm protein in sperm of Diplolaimella sp.; E: Fertilisation of egg in Pontonema vulgaris, capturing an instantaneous process. Abbreviations: ba = basal zone; med = median zone; cor = cortical zone; ep = epicuticle; n sp = nucleus sperm.


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