A quantitative investigation of symplasmic transport in Chara corallina. III. An evaluation of chemical and freeze-substituting techniques in determining the in vivo condition of the plasmodesmata

1975 ◽  
Vol 53 (18) ◽  
pp. 1988-1993 ◽  
Author(s):  
R. A. Fischer ◽  
T. J. MacAlister
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
Room Temperature ◽  
Freeze Substitution ◽  
Chara Corallina ◽  
Chemical Fixation ◽  
Quantitative Investigation ◽  
Internodal Cell ◽  
Symplasmic Transport

We present data on a comparison of plasmodesmata in the nodal complexes of Chara corallina prepared for electron microscopy by chemical fixation of specimens at room temperature and those that have been freeze-substituted. The freeze-substitution technique was applied to intact nodal complexes with the internodal cells on either side intact and showed that most plasmodesmata in the cell walls joining a nodal cell with an internodal cell were free of any occluding substance. We feel that the nonoccluded plasmodesma is the in vivo condition and that previous results using chemical fixation at room temperature where occlusions were reported in Nitella translucens and Chara corallina are artifacts of preparation.

A quantitative investigation of symplasmic transport in Chara corallina. I. Ultrastructure of the nodal complex cell walls

1974 ◽  
Vol 52 (6) ◽  
pp. 1209-1214 ◽  
Author(s):  
R. A. Fischer ◽  
J. Dainty ◽  
M. T. Tyree
Keyword(s):  
Cell Walls ◽  
Pore Diameter ◽  
Chara Corallina ◽  
Central Cell ◽  
Relative Area ◽  
Complex Cell ◽  
Quantitative Investigation ◽  
Peripheral Cell ◽  
Internodal Cell ◽  
Symplasmic Transport

We present a quantitative ultrastructural study of the size and frequency (density distribution) of plasmodesmata in the cell wall in common between the internodal cell and peripheral cell (and central cell) of Chara corallina. In the wall in common between the central cell and internodal cell the relative area occupied by plasmodesmata is 15.3%; the pore diameter (less the plasmalemma) is 118 nm; the length is 1.54 μm, and the frequency is 1.4 × 109 pores/cm2. In the wall in common between the peripheral cell and internodal cell the relative area occupied by the plasmodesmata is 9.6%; the pore diameter is 100 nm; the length is 1.07 μm; and the frequency is 1.2 × 109 pores/cm2. The plasmodesmata have an anastomosing structure like Nitella translucens but the pore diameter and frequency are much greater.

Bacterial envelope profiles revealed by freeze-substitution

1992 ◽  
Vol 50 (1) ◽  
pp. 868-869
Author(s):  
L.L. Graham ◽  
T.J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Chemical Fixation ◽  
Diverse Range ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Conventional Methods ◽  
Section Electron Microscopy ◽  
Better Than

Traditional methods of processing bacteria for thin section electron microscopy rely on chemical fixation and dehydration under conditions which maximize specimen deterioration. Cryotechniques, however, use physical fixation (rapid freezing) and are slowly being recognized as a superior alternative to the more conventional methods. Freeze-substitution is a cryotechnique which combines cryofixation with a gentle chemical fixation and dehydration regimen, yielding specimens amenable to standard embedment procedures and ultramicrotomy. Previous study has shown that freeze-substitution retains the molecular composition of eubacteria better than conventional methods of processing. In this study we extend our observations and show that a simple freeze-substitution protocol reliably preserves the ultrastructure of a diverse range of microorganisms including archaeobacteria and anaerobic eubacteria.

In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles

2018 ◽  
Vol 57 (7) ◽  
pp. 858-863 ◽  
Author(s):  
Júlia de Souza Silveira Valente ◽  
Caroline Quintana Braga ◽  
Carolina Litchina Brasil ◽  
Cristiane Telles Baptista ◽  
Guilherme Fonseca Reis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
In Vitro Assays ◽  
Pythium Insidiosum ◽  
Biogenic Silver Nanoparticles ◽  
Transmission Electron ◽  
Cellular Wall ◽  
Scanning Electron

AbstractPythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 μg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

scholarly journals Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.

1986 ◽  
Vol 103 (6) ◽  
pp. 2541-2550 ◽  
Author(s):  
D W McCurdy ◽  
L H Pratt
Keyword(s):  
Electron Microscopy ◽  
Red Light ◽  
Freeze Substitution ◽  
Immunogold Labeling ◽  
Immunogold Electron Microscopy ◽  
Subcellular Component ◽  
Discrete Structures

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.

scholarly journals Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

2021 ◽  
Author(s):  
Markus Galhuber ◽  
Nadja Kupper ◽  
Gottfried Dohr ◽  
Martin Gauster ◽  
Grazyna Kwapiszewska ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adipose Tissue ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Fresh Tissue ◽  
Simple Method ◽  
Tissue Samples ◽  
Morphometric Analyses ◽  
Brown Adipose ◽  
Mouse Tissues

AbstractPreservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. “biobanking” of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

scholarly journals Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution

2018 ◽  
Vol 66 (12) ◽  
pp. 903-921 ◽  
Author(s):  
Siegfried Reipert ◽  
Helmuth Goldammer ◽  
Christine Richardson ◽  
Martin W. Goldberg ◽  
Timothy J. Hawkins ◽  
...  
Keyword(s):  
Room Temperature ◽  
Low Cost ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
Slow Process ◽  
Safety Issues ◽  
Automated Processing ◽  
Low Cost Manufacturing

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Exhibition of persistent and drug-tolerant L-form habit of Mycobacterium tuberculosis during infection in rats

2008 ◽  
Vol 3 (4) ◽  
pp. 407-416 ◽  
Author(s):  
Nadya Markova ◽  
Lilia Michailova ◽  
Mimi Jourdanova ◽  
Vesselin Kussovski ◽  
Violeta Valcheva ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
Unique Feature ◽  
Peritoneal Lavage ◽  
Liquid Media ◽  
Lowenstein Jensen ◽  
Membrane Bound ◽  
Drug Free ◽  
Primary Drug

AbstractA model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.

Some advantages and uses of cryopreservation techniques for ultrastructural studies in mycology

1991 ◽  
Vol 49 ◽  
pp. 70-71
Author(s):  
Richard E. Edelmann ◽  
Kirk Czymmek ◽  
Karen L. Klomparens
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Freeze Etching ◽  
Cryopreservation Techniques ◽  
Cryo Scanning Electron Microscopy ◽  
Scanning Electron

To date, only limited use has been made of the advanced techniques of cryopreservation for mycological samples, with the exception of the unofficial “lab rat” Saccharomyces cerevisae. However, cyropreservation can offer some distinct advantages over conventional chemical fixation, as well as unique solutions for specific mycological problems. This presentation specifically deals with the utilization of cryo-scanning electron microscopy (cryo-SEM), freeze substitution for transmission electron microscopy (TEM), freeze fracture and freeze etching of mycological samples. Due to the distinctive morphological and biochemical nature of fungi, as compared to plant and animal samples, some significant adaptations to existing published protocols have had to be devised and are presented here.

Cytoskeleton in tobacco plant cells after propane jet freezing and freeze substitution

1990 ◽  
Vol 48 (3) ◽  
pp. 704-705
Author(s):  
Biao Ding ◽  
Robert Turgeon ◽  
M.V. Parthasarathy
Keyword(s):  
Electron Microscopy ◽  
Nicotiana Tabacum ◽  
Sucrose Solution ◽  
Tobacco Plant ◽  
Plant Cells ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Single Plant ◽  
Root Cells ◽  
Cellular Components

Microfilaments in plant cells are very labile and in some cells difficult to preserve by chemical fixation. Single microfilaments are even more labile and are rarely preserved. Cryofixation is so far the best means to preserve these labile cellular components for electron microscopy. However, previous studies only dealt with single plant cells because of the shallow depth of good freezing limited by the technique (plunge freezing). Here we report our results of preserving cytoskeleton in plant leaf and root cells using propane jet freezing and freeze substitution.Tobacco (Nicotiana tabacum L. var. Maryland Mammoth) plants were grown under greenhouse conditions. A developing leaf was cut from a two-month old plant and immersed in a 0.2 M sucrose solution containing 20 mM MES, 20 mM CaCl2 and 20 mM KCl. Samples of 1 mm2 were cut out of the leaf and incubated for 10 to 30 minutes. The leaf samples were then frozen in a commercial propane jet-freezer (Model MF7200, KMC, Tucson, Arizona).

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