Advances in C. elegans Specimen Preparation for TEM Using Microwave and Fast Freeze Techniques

2001 ◽  
Vol 7 (S2) ◽  
pp. 1196-1197
Author(s):  
D.H. Hall ◽  
T. Starich ◽  
J. Shaw ◽  
V. Gobel ◽  
J. Fleming ◽  
...  
Keyword(s):  
Freeze Substitution ◽  
Microwave Energy ◽  
Specimen Preparation ◽  
Genetic Studies ◽  
Larval Cuticle ◽  
C Elegans ◽  
Larval Stages ◽  
Mechanical Cutting ◽  
Fast Freezing

The nematode C. elegans is a simple model for genetic studies of cell and tissue development. There is a need to improve the preservation of embryonic and early larval stages, during which nematode tissues elaborate and separate. However, these stages are particularly resistant to fixation and embedment due an impenetrable eggshell and larval cuticle. Their small size at these ages precludes mechanical cutting, which has been used successfully for immersion fixation of older stages. Here we compare the quality of preservation under three rather different regimes: using laserholes to permeabilize the eggshell during the primary fixation step, using microwave energy to enhance the first fixation step, or using fast freezing and freeze substitution to circumvent the standard immersion procedure. Vancoppenolle et al (2000) have recently demonstrated very good results through enzymatic weakening of the eggshell prior to immersion fixation. Their data are comparable to what we achieve by either the laserhole or microwave methods.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

scholarly journals Functional Characterization of the Adenylyl Cyclase Genesgs-1by Analysis of a Mutational Spectrum inCaenorhabditis elegans

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 133-142 ◽  
Author(s):  
Celine Moorman ◽  
Ronald H A Plasterk
Keyword(s):  
Life Span ◽  
Adenylyl Cyclase ◽  
Neuronal Degeneration ◽  
Functional Characterization ◽  
Adenylyl Cyclases ◽  
Loss Of Function ◽  
C Elegans ◽  
Larval Stages ◽  
Pharyngeal Pumping

AbstractThe sgs-1 (suppressor of activated Gαs) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric Gαs subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

PRE-MEETING SHORT COURSES: JULY 31

2005 ◽  
Vol 11 (I1) ◽  
pp. 23-26
Keyword(s):  
Freeze Substitution ◽  
Specimen Preparation ◽  
Digital Microscopy ◽  
High Pressure Freezing ◽  
Physical Sciences ◽  
Special Events ◽  
Nanoscale Imaging ◽  
Art Exhibit ◽  
Short Courses ◽  
Full Day

Organizers: John Mansfield and Louis KerrAdditional fees required.SC01: Towards Nanoscale Imaging of Anything in VPSEM (including ESEM): From Basics to Current Practices. Full Day: 9:00 AM–5:00 PM, Room 317A.SC02: Image Processing and Analysis. Full Day: 9:00 AM–5:00 PM, Room 317B.SC03: Photoshop for Microscopy and Microanalysis. Full Day: 9:00 AM–5:00 PM, Room 318A.SC04: High Pressure Freezing Cryosectioning of Vitrified Samples for Tomography, and Freeze Substitution. Full Day: 9:00 AM–5:00 PM, Room 318B.SC05: Specimen Preparation for the Physical Sciences. Full Day: 9:00 AM–5:00 PM, Room 319A.SC06: Digital Microscopy and Image Analysis for Materials Characterization. Half Day: 9:00 AM–1:00 PM, Room 319B.SC07: Interpretation of Microstructure. Half-Day: 9:00 AM–1:00 PM, Room 323A.Special Events: Presidential happenings; IMS Henry Clifton Sorby award and lecture; and Art Exhibit.Educational Venues: Microscopic explorations: A workshop; and It's a Family Affair!

scholarly journals Microfluidic-based imaging of complete C. elegans larval development

2021 ◽  
Author(s):  
Simon Berger ◽  
Silvan Spiri ◽  
Andrew deMello ◽  
Alex Hajnal
Keyword(s):  
Imaging Method ◽  
Cover Glass ◽  
Vulval Development ◽  
C Elegans ◽  
Larval Stages ◽  
Seam Cell ◽  
Time Observation ◽  
On Chip ◽  
First Time

Several microfluidic-based methods for long-term C. elegans imaging have been introduced in recent years, allowing real-time observation of previously inaccessible processes. The ex-isting methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method, which allows parallel live-imaging across multiple larval stages, while delivering excellent immobilization and maintaining worm orientation and identity over time. This is achieved by employing an array of microfluidic trap channels carefully tuned to maintain worms in a stable orienta-tion, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition, with the animals remaining free for most of an experiment. Excellent quality images can be ac-quired of multiple worms in parallel, with little impact of the imaging method on worm via-bility or developmental timing. The capabilities of this methodology are demonstrated by observing the hypodermal seam cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate RNAi on-chip, which allows for perturbation of dynamic developmental processes, such as basement membrane breaching during anchor cell invasion.

scholarly journals Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

2022 ◽  
Author(s):  
Martin Schauflinger ◽  
Tim Bergner ◽  
Gregor Neusser ◽  
Christine Kranz ◽  
Clarissa Read
Keyword(s):  
High Pressure ◽  
Potassium Permanganate ◽  
Lipid Membranes ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Block Face ◽  
Critical Aspects

AbstractHigh-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.

1997 ◽  
Vol 200 (10) ◽  
pp. 1509-1514 ◽  
Author(s):  
D L Laughton ◽  
G G Lunt ◽  
A J Wolstenholme
Keyword(s):  
Caenorhabditis Elegans ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Pcr Amplification ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
C Elegans ◽  
Larval Stages ◽  
Major Mode ◽  
Flanking Sequences

Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.

A Fully Automated System for the Preparation of Samples for Cryo-Electron Microscopy

2010 ◽  
Author(s):  
Zachary J. Thompson ◽  
Kevin L. Johnson ◽  
Nicolas Overby ◽  
Jessica I. Chidi ◽  
William K. Pryor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Environmental Control ◽  
Human Interaction ◽  
Automated System ◽  
Specimen Preparation ◽  
Cryo Electron Microscopy ◽  
Early Testing ◽  
High Humidity Environment ◽  
Fully Automated System

The preparation of specimens for cryo-electron microscopy is currently a labor and time intensive process, and the quality of resulting samples is highly dependent on both environmental and procedural factors. Specimens must be applied to sample grids in a high-humidity environment, frozen in liquid ethane, and stored in liquid nitrogen. The combination of cryogenic temperatures and humidity-control mandates the segregation of the humidity-controlled environment from the cryogenic environment. Several devices which automate portions of the specimen preparation process are currently in use; however, these systems still require significant human interaction in order to create viable samples. This paper describes a fully automated system for specimen preparation. The resulting system removes the need for human input during specimen preparation, improves process control, and provides similar levels of environmental control. Early testing shows that the resulting system is capable of manipulating samples in an autonomous manner while providing performance similar to existing systems.

Impact of Cryo Techniques on Cytological Studies of Plant Pathogenic Fungi and their Hosts

2000 ◽  
Vol 6 (S2) ◽  
pp. 674-675 ◽  
Author(s):  
R. J. Howard ◽  
T. M. Bourett ◽  
K. E. Duncan
Keyword(s):  
Electron Microscope ◽  
Freeze Substitution ◽  
Pathogenic Fungi ◽  
Extracellular Matrices ◽  
Direct Result ◽  
Aqueous Environment ◽  
Specimen Preparation ◽  
Plant Pathogenic Fungi ◽  
Chemical Methods ◽  
Transmission Electron

Cryo techniques of specimen preparation have become the standard for cytological studies of biological specimens. From the pioneering work of Feder and Sidman, and Zalokar, the now ubiquitous application of freeze substitution for transmission electron microscope studies of fungi was a direct result of work reported in 1979. Since then, cryopreparative methods have also become the standard for SEM studies and may, for certain purposes, replace conventional methods of fixation for light microscopy as well. Undoubtedly, there are instances where non-cryo methods might be preferred, or where comparisons of results using cryo vs. chemical fixations can provide unique information. On the whole, however, the advantages offered to mycologists and plant pathologists by cryo fixation over any chemical methods are many, and include (a) the opportunity to preserve specimens in a non-aqueous environment, (b) preservation of labile structures such as certain organelles, extracellular matrices, or various cellular content such as ions or that of vacuoles, (c) preservation of cells in a more life-like state,

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