The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

Quantitative x-ray elemental imaging in stimulated single intact skeletal muscle fibers: The fate of calcium in the presence of ryanodine

1988 ◽  
Vol 46 ◽  
pp. 90-91
Author(s):  
J. Sommer ◽  
P. Ingram ◽  
A. LeFurgey ◽  
R. Nassar ◽  
T. High
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Time Course ◽  
Imaging System ◽  
Quantitative Imaging ◽  
Muscle Fibers ◽  
Frog Skeletal Muscle ◽  
X Ray ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried

We are involved in a continuing series of experiments aimed at a complete description,in terms of morphology and quantitative topochemistry, of the time course of spatial distributions of physiologically important elements during excitation-contraction coupling (ECC) at different time intervals (fractions of msec) following electrical stimulation of single, intact frog skeletal muscle fibers. In this present study wg report such distributions for Ca after 1,2 and 3 min of electrical stimulation in the presence of 2x10-4 M ryanodine, an alkaloid that, in time, causes irreversible muscle contractures.Single, intact frog skeletal muscle fibers were quick-frozen, cryosectioned, freeze-substituted and in one case freeze-fractured. The freeze-dried cryosections were subjected to electron probe X-ray microanalysis (EPXMA) in a JEOL 1200EX analytical electron microscope equipped with a Tracor Northern X-ray detector and a fully quantitative imaging system. Both, 64/64 pixel images (ambient temp.), and small raster probes (cold stage,-115 °C) for better statistics, were obtained, each from the same section.

scholarly journals Activity-dependent nuclear translocation and intranuclear distribution of NFATc in adult skeletal muscle fibers

2001 ◽  
Vol 155 (1) ◽  
pp. 27-40 ◽  
Author(s):  
Yewei Liu ◽  
Zoltán Cseresnyés ◽  
William R. Randall ◽  
Martin F. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Nuclear Translocation ◽  
Fiber Type ◽  
Muscle Fibers ◽  
Adult Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Slow Twitch ◽  
Nuclear Foci ◽  
Fast Twitch

TTranscription factor nuclear factor of activated T cells NFATc (NFATc1, NFAT2) may contribute to slow-twitch skeletal muscle fiber type–specific gene expression. Green fluorescence protein (GFP) or FLAG fusion proteins of either wild-type or constitutively active mutant NFATc [NFATc(S→A)] were expressed in cultured adult mouse skeletal muscle fibers from flexor digitorum brevis (predominantly fast-twitch). Unstimulated fibers expressing NFATc(S→A) exhibited a distinct intranuclear pattern of NFATc foci. In unstimulated fibers expressing NFATc–GFP, fluorescence was localized at the sarcomeric z-lines and absent from nuclei. Electrical stimulation using activity patterns typical of slow-twitch muscle, either continuously at 10 Hz or in 5-s trains at 10 Hz every 50 s, caused cyclosporin A–sensitive appearance of fluorescent foci of NFATc–GFP in all nuclei. Fluorescence of nuclear foci increased during the first hour of stimulation and then remained constant during a second hour of stimulation. Kinase inhibitors and ionomycin caused appearance of nuclear foci of NFATc–GFP without electrical stimulation. Nuclear translocation of NFATc–GFP did not occur with either continuous 1 Hz stimulation or with the fast-twitch fiber activity pattern of 0.1-s trains at 50 Hz every 50 s. The stimulation pattern–dependent nuclear translocation of NFATc demonstrated here could thus contribute to fast-twitch to slow-twitch fiber type transformation.

scholarly journals Carbonic anhydrase III in skeletal muscle fibers: an immunocytochemical and biochemical study.

1988 ◽  
Vol 36 (7) ◽  
pp. 775-782 ◽  
Author(s):  
P Frémont ◽  
P M Charest ◽  
C Côté ◽  
P A Rogers
Keyword(s):  
Skeletal Muscle ◽  
Carbonic Anhydrase ◽  
Vastus Lateralis ◽  
Muscle Fibers ◽  
Water Soluble ◽  
Type I ◽  
Fiber Types ◽  
Thin Sections ◽  
Carbonic Anhydrase Iii ◽  
Skeletal Muscle Fibers

The objectives of the present study were to determine if carbonic anhydrase III (CA III) demonstrated a specific association for any particular organelle or structure of the skeletal muscle cell and to quantify the activity and content of this enzyme in different types of skeletal muscle fibers. Ultrastructural localization of CA III in the soleus (SOL), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL), composed of predominantly type I, IIa, and IIb fibers, respectively, was performed using a high-resolution immunocytochemical technique and antibody specific for CA III on ultra-thin sections of skeletal muscle embedded in the water-soluble medium polyvinyl alcohol (PVA). The results indicated a uniform distribution of CA III within the sarcomere. Mitochondria, nuclei, triads, Z-, and M-bands were not specifically labeled. Immunoblotting of washed myofibril preparations did not show any detectable CA III associated with this structure. In addition to quantification of the immunogold labeling, CA III activity and content were assayed in the post-mitochondrial supernatant of the three muscles. In the SOL, these values were found to be 3.6-7.6 times higher than in the DVL. The SVL showed a labeling intensity slightly higher than background level, while the enzyme activity and content were indistinguishable from background levels. We therefore conclude that CA III is randomly distributed in the cytoplasm of the three muscle fiber types and that the relative CA III content and activity in the three muscles studied is SOL greater than DVL greater than SVL approximately equal to 0.

scholarly journals Activity- and Calcineurin-independent Nuclear Shuttling of NFATc1, but Not NFATc3, in Adult Skeletal Muscle Fibers

2006 ◽  
Vol 17 (4) ◽  
pp. 1570-1582 ◽  
Author(s):  
Tiansheng Shen ◽  
Yewei Liu ◽  
Zoltán Cseresnyés ◽  
Arie Hawkins ◽  
William R. Randall ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Nuclear Export ◽  
Muscle Fibers ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Adult Skeletal Muscle ◽  
Casein Kinase 1 ◽  
Nuclear Uptake ◽  
Skeletal Muscle Fibers

The transcription factor NFATc1 may be involved in slow skeletal muscle gene expression. NFATc1 translocates from cytoplasm to nuclei during slow fiber type electrical stimulation of skeletal muscle fibers because of activation of the Ca2+-dependent phosphatase calcineurin, resulting in nuclear factor of activated T-cells (NFAT) dephosphorylation and consequent exposure of its nuclear localization signal. Here, we find that unstimulated adult skeletal muscle fibers exhibit a previously unanticipated nucleocytoplasmic shuttling of NFATc1 without appreciable nuclear accumulation. In resting fibers, the nuclear export inhibitor leptomycin B caused nuclear accumulation of NFATc1 (but not of isoform NFATc3) and formation of NFATc1 intranuclear bodies independent of calcineurin. The rate of nuclear uptake of NFATc1 was 4.6 times lower in resting fibers exposed to leptomycin B than during electrical stimulation. Inhibitors of glycogen synthase kinase and protein kinase A or of casein kinase 1 slowed the decay of nuclear NFATc1 after electrical stimulation, but they did not cause NFATc1 nuclear uptake in unstimulated fibers. We propose that two nuclear translocation pathways, one pathway mediated by calcineurin activation and NFAT dephosphorylation and the other pathway independent of calcineurin and possibly independent of NFAT dephosphorylation, determine the distribution of NFATc1 between cytoplasm and nuclei in adult skeletal muscle.

scholarly journals Freezing of tissue-limits for the autoradiographic localization of diffusible substances.

1979 ◽  
Vol 27 (11) ◽  
pp. 1520-1523 ◽  
Author(s):  
P M Frederik ◽  
W M Busing
Keyword(s):  
Surface Layer ◽  
Electron Microscope ◽  
Crystal Growth ◽  
Freeze Drying ◽  
Crystal Size ◽  
Ice Crystals ◽  
Ice Crystal ◽  
Thin Sections ◽  
Freeze Dried ◽  
Freeze Etching

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.

scholarly journals Elevated nuclear Foxo1 suppresses excitability of skeletal muscle fibers

2013 ◽  
Vol 305 (6) ◽  
pp. C643-C653 ◽  
Author(s):  
Erick O. Hernández-Ochoa ◽  
Tova Neustadt Schachter ◽  
Martin F. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Atrophy ◽  
Action Potentials ◽  
Muscle Fibers ◽  
Calcium Transients ◽  
Skeletal Muscle Atrophy ◽  
Nuclear Activity ◽  
Ec Coupling ◽  
Skeletal Muscle Fibers

Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.

Simplified Sample Embedding and Polishing Methods for Preparing Hydrophilic, Fragile, or Solvent-Susceptible Materials for Thin Sections for Microscopic Analyses

2019 ◽  
Vol 25 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Masato Ueshima ◽  
Hirofumi Sakanakura
Keyword(s):  
Thin Section ◽  
Crystal Formation ◽  
Polarization Microscopy ◽  
Ice Crystal ◽  
Electron Backscattered Diffraction ◽  
Preparation Methods ◽  
Thin Sections ◽  
X Ray ◽  
Flat Surfaces ◽  
Freeze Dried

AbstractIn the preparation of thin sections for microscopy, embedding and polishing processes in particular can change the composition and morphologies of samples. Soils and ashes are very fragile and solvent-susceptible, and appropriate sample preparation procedures have not been well-established. To improve the existing preparation methods and make them easier and faster, we embedded freeze-dried blocks, polished, and then examined these thin-section samples using polarization microscopy, laser microscopy, and field emission scanning electron microscopy with energy-dispersive X-ray spectrometry, and electron backscattered diffraction (EBSD). Appropriate thin-section samples can be prepared by: (1) rinsing with acetone and then embedding with Spurr resin along with repeated evacuation and ventilation, rather than conventional dehydration/replacement; (2) polishing using silicon carbide paper and diamond slurries, and then wiping with a cloth and a synthetic oil; and (3) slightly rinsing with 100% ethanol to remove the oil. The preparation method minimized contamination and pores, and showed flat surfaces and sometimes EBSD patterns. Freeze-drying has been claimed to cause the development of cracks due to ice crystal formation upon freezing, however, our method not only overcomes such problems for microscopic observation but saves substantial time, taking only 2 days in total to process a specimen, and requiring less than 1 g of resin and ~1 g of sample.

scholarly journals Effects of Immersion Freezing on Ice Crystal Formation and the Protein Properties of Snakehead (Channa argus)

Foods ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 411
Author(s):  
Shulai Liu ◽  
Xiaohong Zeng ◽  
Zhenyu Zhang ◽  
Guanyu Long ◽  
Fei Lyu ◽  
...  
Keyword(s):  
Food Preservation ◽  
Ice Crystals ◽  
Random Coil ◽  
Maximum Temperature ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Protein Properties ◽  
Immersion Freezing ◽  
Ft Ir ◽  
Ice Crystal Formation

This study aimed to evaluate the effect of immersion freezing (IF) at different temperatures on ice crystal formation and protein properties in fish muscle. Snakehead blocks were frozen by IF at −20, −30, and −40 °C, and conventional air freezing (AF) at −20 °C. The size of ice crystals in the frozen samples was evaluated using Image J software. Changes in protein properties were analyzed by Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Snakehead blocks frozen using IF contained smaller ice crystals and better microstructures, especially at lower temperatures. The mean cross-sectional areas of ice crystals formed in the frozen samples were 308.8, 142.4, and 86.5 μm2 for IF treatments at −20, −30, and −40 °C, respectively, and 939.6 μm2 for the AF treatment. The FT-IR results show that protein aggregation in the frozen fish blocks was manifested by a decrease in α-helices connected to the increased random coil fraction. The DSC results show that samples prepared by IF had a higher denaturation enthalpy (∆H) and denaturation maximum temperature (Tmax) than those prepared by AF. These results confirm that IF generated a larger number of smaller ice crystals, which is conducive to food preservation.

scholarly journals Gerta Vrbová, a guide and a friend for a generation of neuro-myologists – Her scientific legacies and relations with colleagues

2021 ◽  
Author(s):  
Ugo Carraro
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Neuromuscular Disorders ◽  
Muscle Fibers ◽  
Low Frequency ◽  
Scientific Heritage ◽  
Adult Muscle ◽  
Skeletal Muscle Fibers ◽  
Different Types ◽  
The Many

Gerta Sidonová - Vrbová, (Trnava, Slovakia, November 28, 1926 - London, UK, October 2, 2020) has been a key neuroscientist, who for almost half a century has contributed important findings and hypotheses on the relationships between motoneurons and skeletal muscle fibers, in particular on the differentiation and extent of plasticity of the peculiar characteristics of the different types of fibers present in mammalian muscles. This issue, Ejtm 31 (1), 2021, opens with the personal obituary authored by Dirk Pette, who remember his lifelong collaboration with Gerta, describing the many molecular and metabolic events that occur by changing the pattern of activation of adult muscle fibers through neuromuscular low frequency electrical stimulation. To honor the many scientific legacies of Gerta Vrbová and her impact on a generation of researchers studying myology and managements of neuromuscular disorders I add here additional examples of Gerta’s scientific heritage and of her relations with colleagues.

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