Efficient fluorescence recovery using antifade reagents in correlative light and electron microscopy

Microscopy ◽  
2019 ◽  
Vol 68 (5) ◽  
pp. 417-421
Author(s):  
Kiminori Toyooka ◽  
Naeko Shinozaki-Narikawa
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Ultrastructural Level ◽  
Fluorescence Decay ◽  
Fluorescence Recovery ◽  
Recovery Method ◽  
Thin Sections ◽  
Electron Microscopies ◽  
Green Fluorescent

Abstract Correlative light and electron microscopy (CLEM) enables ultrastructural-level analysis of fluorescence-labeled proteins by combining images obtained from both fluorescence and electron microscopies. A technical challenge with the CLEM method is the effective detection of fluorescence from samples embedded in resins, which generally cause fluorescence decay. To overcome this issue, we developed a method for fluorescence recovery of green fluorescent protein (GFP) in resin-embedded semi-thin sections using commercially available antifade reagents. By applying this method, we successfully obtained CLEM images using field-emission scanning electron microscopy with moderately enhanced GFP signals, demonstrating the efficacy of this simple fluorescence recovery method.

Detection and localization of the endophyte Undifilum oxytropis in locoweed tissues

Botany ◽  
2012 ◽  
Vol 90 (12) ◽  
pp. 1229-1236 ◽  
Author(s):  
Roxanna Reyna ◽  
Peter Cooke ◽  
Daniel Grum ◽  
Daniel Cook ◽  
Rebecca Creamer
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Growth Patterns ◽  
Host Cells ◽  
Economic Losses ◽  
Thin Sections ◽  
Oxytropis Sericea ◽  
Pathogenic Interaction ◽  
Green Fluorescent ◽  
Detection And Localization

Poisoning of livestock owing to grazing on locoweeds results in significant economic losses in the western United States. Some Oxytropis spp. locoweeds contain a seed-transmitted endophytic fungus, Undifilum oxytropis, which produces the toxic alkaloid swainsonine. We sought to localize and characterize growth patterns of the fungus within leaves and petioles of Oxytropis lambertii Pursh and Oxytropis sericea Nutt. to help define the types of interactions between the fungus and its hosts. Vegetative hyphae were observed within locoweed tissues using integrated imaging. Topographical images from scanning electron microscopy revealed the presence of the endophyte in the pith tissue of petioles. The fungus was identified between plant cells but did not appear to penetrate host cells. Transmission electron microscopy images of thin sections revealed that hyphae were closely associated with host cell walls. Oxytropis sericea was innoculated with green fluorescent protein-transformed U. oxytropis and observed by confocal microscopy, confirming the presence of the endophyte hyphae in leaves and petioles. The fungus was identified only in the pith of petioles using fluorescence and in the vascular bundle throughout extracellular spaces in leaves. These results revealed no signs of a pathogenic interaction between plant and fungus and support the hypothesis of a mutualistic or commensal relationship.

Visualizing Green Fluorescent Protein and Fluorescence Associated with a Gold Conjugate in Thin Sections with Correlative Confocal and Electron Microscopy

2004 ◽  
Vol 10 (S02) ◽  
pp. 156-157 ◽  
Author(s):  
Paul. A Sims ◽  
Jeff Hardin
Keyword(s):  
Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Thin Sections ◽  
Gold Conjugate ◽  
Green Fluorescent ◽  
Confocal And Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

scholarly journals Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy

2010 ◽  
Vol 16 (6) ◽  
pp. 747-754 ◽  
Author(s):  
Hugo H. Hanson ◽  
James E. Reilly ◽  
Rebecca Lee ◽  
William G. Janssen ◽  
Greg R. Phillips
Keyword(s):  
Electron Microscopy ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Laser Scanning Confocal Microscopy ◽  
Step Method ◽  
Cell Monolayers ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron ◽  
Green Fluorescent

AbstractCorrelative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle

1994 ◽  
Vol 107 (8) ◽  
pp. 2191-2202 ◽  
Author(s):  
P. Hozak ◽  
D.A. Jackson ◽  
P.R. Cook
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
S Phase ◽  
Nuclear Bodies ◽  
Nuclear Antigen ◽  
Thin Sections ◽  
Permeabilized Cells ◽  
Ultrastructural Characterization ◽  
Replication Sites ◽  
Proliferating Cell

Sites of replication in synchronized HeLa cells were visualized by light and electron microscopy; cells were permeabilized and incubated with biotin-16-dUTP, and incorporation sites were immunolabelled. Electron microscopy of thick resinless sections from which approximately 90% chromatin had been removed showed that most DNA synthesis occurs in specific dense structures (replication factories) attached to a diffuse nucleoskeleton. These factories appear at the end of G1-phase and quickly become active; as S-phase progresses, they increase in size and decrease in number like sites of incorporation seen by light microscopy. Electron microscopy of conventional thin sections proved that these factories are a subset of nuclear bodies; they changed in the same characteristic way and contained DNA polymerase alpha and proliferating cell nuclear antigen. As replication factories can be observed and labelled in non-permeabilized cells, they cannot be aggregation artifacts. Some replication occurs outside factories at discrete sites on the diffuse skeleton; it becomes significant by mid S-phase and later becomes concentrated beneath the lamina.

Cytology and ultrastructure of Thanatephorus cucumeris and related taxa of the Rhizoctonia complex

1977 ◽  
Vol 55 (18) ◽  
pp. 2419-2436 ◽  
Author(s):  
C. C. Tu ◽  
James W. Kimbrough ◽  
H. C. Aldrich
Keyword(s):  
Electron Microscopy ◽  
Light Microscope ◽  
Light And Electron Microscopy ◽  
Middle Layer ◽  
Ultrastructural Level ◽  
Aniline Blue ◽  
Diploid Nucleus ◽  
Thanatephorus Cucumeris ◽  
Light Microscope Level ◽  
Endoplasmic Reticula

Cytological studies on the vegetative hyphae of members of the Rhizoctonia complex and basidial structures of Thanatephorus cucumeris were performed with light and electron microscopy. Vegetative cells of Thanatephorus and Waitea proved to be multinucleate, whereas those of Uthatobasidium, Ceratobasidium, Athelia. and Botryobasidium are binucleate.Dolipore septa of Thanatephorus, Waitea, Uthatobasidium, and Ceratobasidium are visible with the light microscope when stained with aniline blue in glycerine. Ultrastructurally, pore caps in these genera consisted of two-layered unit membranes, forming cisternae with an electron-dense middle layer. Dolipore septa of Athelia (S. rolfsii) and Botryobasidium are not visible in aniline blue at the light microscope level. At the ultrastructural level, there was an additional cisternal membrane making up a pore cap of three membranes. The fine structure of nuclei, mitochondria, endoplasmic reticula, vacuoles, and other organelles in the basidial structures of T. cucumeris was essentially the same as in other basidiomycetes.Karyogamy of two haploid nuclei occurs in the young basidia of T. cucumeris. The nuclear envelopes of both haploid nuclei break at their adjacent sides and fuse to form a diploid nucleus. After a short interphase, meiosis occurs. No leptotene was observed at prophase I, but a synaptinemal complex was evident and six pairs of chromosomes were observed throughout pachytene, diplotene, and diakinesis. The nuclear envelope disappears at metaphase I and a spindle appears. The second meiotic division is equational. Most of the mature and discharged spores are uninucleate.

scholarly journals Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

1988 ◽  
Vol 36 (7) ◽  
pp. 717-727 ◽  
Author(s):  
S J Hagen ◽  
J S Trier
Keyword(s):  
Electron Microscopy ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Light And Electron Microscopy ◽  
Basal Membrane ◽  
Ground Substance ◽  
Structural Protein ◽  
Thin Sections ◽  
Filament Bundles ◽  
Lowicryl K4m

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

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