Cryotechniques in Biological Electron Microscopy

doi:10.1007/978-3-642-72815-0

Cited by 122 publications

(2 citation statements)

References 236 publications

(353 reference statements)

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“…Cryo-fixation circumvents this problem and preserves the cytoplasm in a fraction of a second; however, flawless vitrification is difficult to achieve except for the outermost micrometers (e.g., 10 μm) of tissues; in deeper layers of cells and tissues, ice crystal damage occurs. The technique of highpressure freeze fixation followed by freeze substitution greatly increases the depth and extent of high-quality fixation as described by Knoll et al (1987), Moor (1987), Studer et al (2001), andMcDonald et al (2007) and since then yielded some excellent results in plant cells (e.g., Donohoe et al 2007;Wilson and Bacic 2012;Karahara and Kang 2014;Gergely et al 2018) and animal cells (e.g., Hess et al 2018).…”

Section: Lessons Learned From Em and Quality Of The Fixationmentioning

confidence: 99%

Gland cell responses to feeding in Drosera capensis, a carnivorous plant

et al. 2021

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Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

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Section: Lessons Learned From Em and Quality Of The Fixationmentioning

confidence: 99%

Gland cell responses to feeding in Drosera capensis, a carnivorous plant

et al. 2021

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“…Nevertheless, whenever the development of minute structures such as sensilla, ommatidia, glands, synaptic connectivity in nervous systems and, in general, differentiation processes on the cellular level are to be studied, the TEM is the right method at hand (e.g., [74][75][76] for sensilla in general, and [77,78] for crustacean larvae). Two main preparation techniques are used in TEM studies, the first is based on chemical fixation and represents the standard (see Table 1 in the companion paper [8]), whereas the second is based on cryofixation and is more sophisticated (surveys [72,73,[79][80][81]). Cryotechniques circumvent some of the artefacts of chemical fixation, but are mainly useful for very smallsized specimens or objects in the 10-µm range.…”

Section: An Oldie But Still a Goldie-classical Histology Introductionmentioning

confidence: 99%

Methods to study organogenesis in decapod crustacean larvae II: analysing cells and tissues

et al. 2021

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Cells and tissues form the bewildering diversity of crustacean larval organ systems which are necessary for these organisms to autonomously survive in the plankton. For the developmental biologist, decapod crustaceans provide the fascinating opportunity to analyse how the adult organism unfolds from organ Anlagen compressed into a miniature larva in the sub-millimetre range. This publication is the second part of our survey of methods to study organogenesis in decapod crustacean larvae. In a companion paper, we have already described the techniques for culturing larvae in the laboratory and dissecting and chemically fixing their tissues for histological analyses. Here, we review various classical and more modern imaging techniques suitable for analyses of eidonomy, anatomy, and morphogenetic changes within decapod larval development, and protocols including many tips and tricks for successful research are provided. The methods cover reflected-light-based methods, autofluorescence-based imaging, scanning electron microscopy, usage of specific fluorescence markers, classical histology (paraffin, semithin and ultrathin sectioning combined with light and electron microscopy), X-ray microscopy (µCT), immunohistochemistry and usage of in vivo markers. For each method, we report our personal experience and give estimations of the method’s research possibilities, the effort needed, costs and provide an outlook for future directions of research.

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