Low‐temperature scanning electron microscopy in biology

Read, Nick D.; Jeffree, C. E.

doi:10.1111/j.1365-2818.1991.tb03073.x

Cited by 76 publications

(42 citation statements)

References 51 publications

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“…A review of the literature revealed that fusion between spore germlings is common, and over 20 species were found to produce structures similar to CATs (35,36). Köhler (24) first showed fusion between germlings of Neurospora spp.The initial aim of the present study was to analyze the morphogenetic process of fusion between conidia and conidial germlings of N. crassa by using confocal live-cell imaging (9, 16) and low-temperature scanning electron microscopy of frozen-hydrated samples (33,34). We found that the three types of conidia (macroconidia, microconidia, and arthroconidia) produced by N. crassa (40) form CATs that are morphologically distinct and different in behavior from germ tubes.…”

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Cell Biology of Conidial Anastomosis Tubes in Neurospora crassa

Arlt²,

et al. 2005

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Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.Hyphal fusion (anastomosis) is commonly undergone at two stages during the development of the vegetative colony of a filamentous fungus. It occurs initially between spores or spore germlings during the early stages of colony establishment and subsequently between hyphae located behind leading hyphae of the peripheral growth zone of the mycelium. Among other proposed roles, vegetative hyphal fusion is believed to facilitate communication and the transport of water and nutrients within the colony by producing a complex interconnected hyphal network (14).Neurospora crassa is proving to be an excellent model system in which to analyze vegetative hyphal fusion (12,14). Live-cell analysis of hyphal fusion in mature colonies of N. crassa demonstrated that specialized fusion hyphae exhibit positive tropisms by growing (homing) toward each other, and their close vicinity to other hyphae can induce the formation of further fusion hyphae (15). Different stages in the homing and fusion process were defined, and this has been summarized by Glass et al. (14). The nature of the diffusible chemoattractant molecules responsible for these tropisms is unknown (15). A number of mutants unable to undergo hyphal fusion have been isolated. Mutants disrupted in the mak-2 and nrc-1 genes encode a mitogen-activated protein (MAP) kinase and a MAP kinase kinase kinase, respectively. These prote...

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Cell Biology of Conidial Anastomosis Tubes in Neurospora crassa

Arlt²,

et al. 2005

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“…Glomus intraradices, T. harzianum and R. solani) on autotrophic potato plantlets as a single host. For the three fungi, the hyphal extension, colonization of the substrate, root infection/colonization and reproduction structures were described under autotrophic in vitro culture conditions (Voets et al, 2005), combining the video camera imaging (De la Providencia et al, 2005) and the cryo-electron scanning microscope (cryo-SEM) (Read and Jeffree, 1991).…”

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Coupling autotrophic in vitro plant cultivation system to scanning electron microscope to study plant-fungal interactions

Jaeger

Decock

Declerck

et al. 2010

Span. j. agric. res.

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The interactions of plants with pathogens and beneficial micro-organisms have been seldom compared on the same host and under strict controlled autotrophic in vitro culture conditions. Here, the life cycle of two plant beneficial (Glomus sp. MUCL 41833 and Trichoderma harzianum) and one plant pathogen (Rhizoctonia solani) fungi were described on potato (Solanum tuberosum) plantlets under autotrophic in vitro culture conditions using video camera imaging and the scanning electron microscope (SEM). (i) The colony developmental pattern of the extraradical mycelium within the substrate, (ii) the reproduction structures and (iii) the three-dimensional spatial arrangements of the fungal hyphae within the potato root cells were successfully visualized, monitored and described. The combination of the autotrophic in vitro culture system and SEM represent a powerful tool for improving our knowledge on the dynamics of plant-fungal interactions.

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“…Thus, images with good representation of three-dimensional shape of samples can be achieved [Wikipedia]. SEMs equipped with cold stage for cryo-microscopy can be used for cryofixation as low temperature SEM [Read & Jeffree, 1991]. Cryo-fixed specimens can also be cryo-fractured under vacuum to reveal internal structures [Faulkner et al, 2008].…”

Section: Introductionmentioning

confidence: 99%

“…Glutaraldehyde and osmium tetroxide are often used to crosslink protein molecules and to preserve lipids, respectively. This step is followed by post-fixation with osmium tetroxide (OsO4) [Read & Jeffree, 1991]. A buffer containing sodium cacodylate, Sucrose and 2-Mercaptoethanol is recommended if precipitation is visible in final sections.…”

Section: Introductionmentioning

confidence: 99%

“…Fixation is a general term used to describe the process of preserving a sample at a moment in time and to prevent further deterioration so that it appears as close as possible to what it would be like in the living state, although it is now dead. Fixation is performed by incubation of samples in a solution of a buffered chemical aldehyde-based fixative, such as glutaraldehyde, paraformaldehyde, or formaldehyde or combinations of these [Read & Jeffree, 1991, Karnovsky, 1965, Kiernan, 2000. For primary fixation, glutaraldehyde buffered (pH 7.4) in sodium cacodylate, Sucrose or phosphate buffers (PBS, HEPES, and PIPES), are used.…”

Section: Introductionmentioning

confidence: 99%

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