scholarly journals FluoroNanogold Is a Bifunctional Immunoprobe for Correlative Fluorescence and Electron Microscopy

2000 ◽  
Vol 48 (4) ◽  
pp. 481-485 ◽  
Author(s):  
Toshihiro Takizawa ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Particle ◽  
High Spatial Resolution ◽  
Human Neutrophils ◽  
Secondary Antibody ◽  
Marker Protein ◽  
Cytoplasmic Granules ◽  
Fluorescence And Electron Microscopy ◽  
Ultrathin Cryosections

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for highresolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.

Fluoronanogold as a Probe for High Resolution Correlation Between ImmunoFluorescence and Electron Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 476-477
Author(s):  
T. Takizawa ◽  
J. M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Immunofluorescence Microscopy ◽  
Human Neutrophils ◽  
Secondary Antibody ◽  
Marker Protein ◽  
Fab Fragment ◽  
Ultrathin Cryosections

[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.

scholarly journals Efficient Immunocytochemical Labeling of Leukocyte Microtubules with FluoroNanogold: An Important Tool for Correlative Microscopy

1997 ◽  
Vol 45 (5) ◽  
pp. 631-642 ◽  
Author(s):  
John M. Robinson ◽  
Dale D. Vandré
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Particle ◽  
Model System ◽  
The Other ◽  
Secondary Antibody ◽  
Correlative Microscopy ◽  
Silver Enhancement ◽  
Fab Fragment ◽  
Gold Particles

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

scholarly journals Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle

1998 ◽  
Vol 46 (10) ◽  
pp. 1097-1102 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Kouki Suzuki ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Imaging Techniques ◽  
Human Neutrophils ◽  
Fluorescence Signal ◽  
Correlative Microscopy ◽  
Reporter System ◽  
Fluorescence And Electron Microscopy ◽  
Specific Granules ◽  
Ultrathin Cryosections ◽  
Proof Of Principle

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for highresolution correlation between immunofluorescence and electron microscopy.

scholarly journals Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy.

1985 ◽  
Vol 33 (10) ◽  
pp. 995-1000 ◽  
Author(s):  
R B Alexander ◽  
W B Isaacs ◽  
E R Barrack
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Intermediate Filaments ◽  
Colloidal Gold ◽  
Microscopic Level ◽  
Secondary Antibody ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Cell Monolayers ◽  
Adenocarcinoma Cells

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

Bacterial and Mineral Elements in an Arctic Biofilm: A Correlative Study Using Fluorescence and Electron Microscopy

2010 ◽  
Vol 16 (2) ◽  
pp. 153-165 ◽  
Author(s):  
Samuel Clarke ◽  
Randall E. Mielke ◽  
Andrea Neal ◽  
Patricia Holden ◽  
Jay L. Nadeau
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Close Association ◽  
Environmental Scanning Electron Microscopy ◽  
High Arctic ◽  
Mineral Elements ◽  
Environmental Scanning ◽  
Gram Negative ◽  
Fluorescence And Electron Microscopy ◽  
Scanning Transmission

AbstractFew simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

scholarly journals Ultrathin Cryosections: An Important Tool for Immunofluorescence and Correlative Microscopy1

2003 ◽  
Vol 51 (6) ◽  
pp. 707-714 ◽  
Author(s):  
Toshihiro Takizawa ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Ultrathin Section ◽  
Detection System ◽  
Placental Tissue ◽  
Counting Procedure ◽  
Fluorescence And Electron Microscopy ◽  
Ultrathin Cryosections ◽  
High Degree ◽  
Number Of Particles

Here we show that ultrathin cryosections of placental tissue can be used as a substrate in immunofluorescence experiments. A high degree of spatial resolution can be achieved in these preparations because there is essentially no out-of-focus fluorescence. Therefore, immunofluorescence microscopy using ultrathin cryosections provides a very useful method for determining the precise subcellular localization of antigens in tissues. In addition, ultrathin cryosections of placenta also serve as a substrate for correlative immunofluorescence and immunoelectron microscopy using FluoroNanogold as the detection system. In correlative microscopy, the exact same structures in the same ultrathin section were observed by both fluorescence and electron microscopy. Using a particle counting procedure and electron microscopy, we compared the labeling obtained with colloidal gold and FluoroNanogold and found a higher number of particles with silver-enhanced FluoroNanogold than with colloidal gold.

Immunolabeling for Correlative Light and Electron Microscopy on Ultrathin Cryosections

2008 ◽  
Vol 14 (2) ◽  
pp. 159-165 ◽  
Author(s):  
Irawati K. Kandela ◽  
Reiner Bleher ◽  
Ralph M. Albrecht
Keyword(s):  
Electron Microscopy ◽  
Spatial Resolution ◽  
Light And Electron Microscopy ◽  
Primary Antibody ◽  
Fluorescence Signal ◽  
Data Sets ◽  
Secondary Antibody ◽  
Transmission Electron ◽  
Sufficient Distance ◽  
Ultrathin Cryosections

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.

A web-like network of type vi collagen in human skin is revealed by immuno-electron microscopy

1988 ◽  
Vol 46 ◽  
pp. 382-383
Author(s):  
Douglas R. Keene ◽  
Robert W. Glanville ◽  
Eva Engvall
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Human Skin ◽  
Basement Membranes ◽  
Primary Antibody ◽  
Fat Cells ◽  
Secondary Antibody ◽  
Type Vi Collagen ◽  
Dermal Matrix ◽  
Immuno Electron Microscopy

A mouse monoclonal antibody (5C6) prepared against human type VI collagen (1) has been used in this study to immunolocalize type VI collagen in human skin. The enbloc method used involves exposing whole tissue pieces to primary antibody and 5 nm gold conjugated secondary antibody before fixation, and has been described in detail elsewhere (2).Biopsies were taken from individuals ranging in age from neonate to 65 years old. By immuno-electron microscopy, type VI collagen is found to be distributed as a fine branching network closely associated with (but not attached to) banded collagen fibrils containing types I and III collagen (Fig. 1). It appears to enwrap fibers, to weave between individual fibrils within a fiber, and to span the distance separating fibers, creating a “web-like network” which entraps fibers within deep papillary and reticular dermal layers (Fig. 2). Relative to that in the dermal matrix, the concentration of type VI collagen is higher around endothelial basement membranes limiting the outer boundaries of nerves, capillaries, and fat cells (Fig. 3).

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