Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

Freeze substitution studies on cultured human fibroblasts

1989 ◽  
Vol 47 ◽  
pp. 1008-1009
Author(s):  
Julian P. Heath ◽  
Donna Turner
Keyword(s):  
Osmium Tetroxide ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Lung Fibroblasts ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Cultured Human Fibroblasts

We are using rapid freezing and freeze substitution to study the three dimensional organisation of membrane systems and cytoskeletal filaments in motile fibroblasts. This study has two objectives: first, to provide material for structural and immunocytochemical analysis of membrane-cytoskeletal interactions in cells that have been preserved with minimum artefact (1,2,3) and second, to refine and develop existing rapid freezing and freeze substitution techniques to allow for the study of single cells that have been experimentally manipulated and observed by digital video microscopy before fixation.The cells used were human lung fibroblasts (IMR90) either growing on Lux Thermanox coverslips or as pelleted suspensions. The cells were slam frozen on a Med-Vac Cryo Press against a liquid nitrogen cooled copper block. Coverslips were trimmed to 2 x 2 mm in size, excess fluid was drained off, and they were placed on top of a 1mm thick gelatin cushion on an aluminium planchette. For cell suspensions, 3 ul was placed on top of the gelatin cushion. Frozen samples were placed in acetone containing 1% osmium tetroxide for 72 hours at 192 K, wanned to 253 K for 4 hours, and then brought to room temperature. The samples were rinsed in acetone and embedded in Spurr’s resin. Thin sections were cut on a RMC6000 ultramicrotome, stained in uranyl acetate and Reynolds' lead citrate and photographed on a Philips EM410 electron microscope at 60 keV.

Freeze-substitution of rye grass and ragweed pollen grains

1990 ◽  
Vol 48 (3) ◽  
pp. 686-687
Author(s):  
Liza B. Martinez ◽  
Susan M. Wick
Keyword(s):  
Cell Function ◽  
Pollen Grains ◽  
Freeze Substitution ◽  
Cell Types ◽  
Rapid Freezing ◽  
Rye Grass ◽  
Acrylic Resins ◽  
Allergenic Proteins ◽  
Cold Embedding ◽  
Structural Preservation

Rapid freezing and freeze-substitution have been employed as alternatives to chemical fixation because of the improved structural preservation obtained in various cell types. This has been attributed to biomolecular immobilization derived from the extremely rapid arrest of cell function. These methods allow the elimination of conventionally used fixatives, which may have denaturing or “masking” effects on proteins. Thus, this makes them ideal techniques for immunocytochemistry, in which preservation of both ultrastructure and antigenicity are important. These procedures are also compatible with cold embedding acrylic resins which are known to increase sensitivity in immunolabelling.This study reveals how rapid freezing and freeze-substitution may prove to be useful in the study of the mobile allergenic proteins of rye grass and ragweed. Most studies have relied on the use of osmium tetroxide to achieve the necessary ultrastructural detail in pollen whereas those that omitted it have had to contend with poor overall preservation.

Cell-sorting in aggregates of Dictyostelium discoideum

1999 ◽  
Vol 112 (22) ◽  
pp. 3923-3929 ◽  
Author(s):  
A. Nicol ◽  
W. Rappel ◽  
H. Levine ◽  
W.F. Loomis
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Three Dimensional ◽  
Cell Movement ◽  
Time Lapse ◽  
Cell Types ◽  
Cell Type ◽  
Prespore Cell ◽  
Green Fluorescent

When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

scholarly journals Evidence for "twisted plane" undulations in golden hamster sperm tails

1977 ◽  
Vol 75 (3) ◽  
pp. 851-865 ◽  
Author(s):  
DM Woolley
Keyword(s):  
Electron Microscopy ◽  
Low Temperature ◽  
Ethylene Glycol ◽  
Golden Hamster ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
The Third ◽  
Working Hypothesis ◽  
Angular Displacements

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.

Freeze substitution fixation of fungal reproductive structures and spores

1989 ◽  
Vol 47 ◽  
pp. 990-991
Author(s):  
C. W. Mims ◽  
E. A. Richardson
Keyword(s):  
Plant Pathogens ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Dialysis Membrane ◽  
Razor Blade ◽  
Thin Sections ◽  
Reproductive Structures ◽  
Dry Down ◽  
Diamond Knife

The advantages of freeze substitution fixation over conventional chemical fixation for preservation of ultrastructural details in fungi have been discussed by various authors. As most ascomycetes, basidiomycetes and deuteromycetes do not fix well using conventional chemical fixation protocols, freeze substitution has attracted the attention of many individuals interested in fungal ultrastructure. Thus far most workers using this technique on fungi have concentrated on thin walled somatic hyphae. However, in our laboratory we have experimented with the use of freeze substitution on a variety of fungal reproductive structures and spores with promising results.Here we present data on freeze substituted samples of sporangia of the zygomycete Umbellopsis vinacea, basidia of Exobasidium camelliae var. gracilis, developing teliospores of the smut Sporisorium sorghi, germinating teliospores of the rust Gymnosporangium clavipes, germinating conidia of the deuteromycete Cercosporidium personatum, and developing ascospores of Ascodesmis nigricans.Spores of G. clavipes and C. personatum were deposited on moist pieces of sterile dialysis membrane where they hydrated and germinated. Asci of A. nigricans developed on pieces of dialysis membrane lying on nutrient agar plates. U. vinacea was cultured on small pieces of agar-coated wire. In the plant pathogens E. camelliae var. gracilis and S. sorghi, a razor blade was used to remove smal1 pieces of infected host issue. All samples were plunged directly into liquid propane and processed for study according to Hoch.l Samples on dialysis membrane were flat embedded. Serial thin sections were cut using a diamond knife, collected on slot grids, and allowed to dry down onto Formvar coated aluminum racks. Sections were post stained with uranyl acetate and lead citrate.

scholarly journals Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

2013 ◽  
Vol 34 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Kazuto Masamoto ◽  
Hiroyuki Takuwa ◽  
Chie Seki ◽  
Junko Taniguchi ◽  
Yoshiaki Itoh ◽  
...  
Keyword(s):  
Adult Mouse ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Mouse Cortex ◽  
Hypoxia Adaptation ◽  
Green Fluorescent

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14  μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60  μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

Developmental derivation of vocal fold mucosa from human induced pluripotent stem cells

2019 ◽  
Author(s):  
Vlasta Lungova ◽  
Susan Thibeault
Keyword(s):  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Vocal Fold ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Stratified Squamous Epithelium ◽  
Three Dimensional Models ◽  
Induced Pluripotent ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa.

Ultrastructure of the Infection of Poinsettia by Oidium SP. using High Pressure Freezing and Freeze Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 690-691
Author(s):  
G. J. Celio ◽  
E. A. Richardson ◽  
C. W. Mims
Keyword(s):  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Euphorbia Pulcherrima ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Transmission Electron ◽  
Dextran Solution ◽  
Leaf Disks ◽  
Diamond Knife

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

scholarly journals Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9102-9123 ◽  
Author(s):  
Shaohui Huang ◽  
Larry Lifshitz ◽  
Varsha Patki-Kamath ◽  
Richard Tuft ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Ph Domain ◽  
Membrane Ruffling ◽  
Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

Sign in / Sign up

Export Citation Format

Share Document