ARTICLE |
Correspondence to: John M. Robinson, Dept. Cell Biology, Neurobiology, and Anatomy, Ohio State U., 4072 Graves Hall, 333 West 10th, Columbus, OH 43210.
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Summary |
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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. (J Histochem Cytochem 45:631-642, 1997)
Key Words: FluoroNanogold, immunocytochemistry, correlative microscopy, fluorescence microscopy, confocal microscopy, electron microscopy, microtubules, neutrophils, monocytes
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Introduction |
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Imaging of cells and subcellular structures continues to be very important in biological investigations. There are often situations in which it is desirable to examine specimens by multiple imaging modalities. Correlative microscopy, in which a single sample can be examined by two or more imaging techniques (e.g., fluorescence and electron microscopy) or when multiple structures or molecules are viewed with the same imaging procedure (e.g., fluorescence microscopy) have been important in many studies related to cell structure and function (e.g.,
The application of immunocytochemistry in cell and developmental biology as well as in immunopathology has been extremely important in defining the distribution of molecules in both spatial and temporal domains. Two of the major reporter systems used in immunocytochemistry have been the labeling of immunoprobes with fluorescent and particulate markers. The use of fluorescent immunoprobes offers the advantages afforded by high-resolution optical and confocal microscopes for their detection as well as the availability of many fluorochromes with different spectral properties for multiple labeling. Particulate probes, especially colloidal gold, have permitted high-resolution detection of molecules at the ultrastructural level. The availability of colloidal gold particles in different sizes has allowed the detection of more than one type of molecule in the same cell.
Although colloidal gold has been invaluable for ultrastructural immunocytochemistry, several studies have shown that there is an inverse relationship between the size of colloidal gold particles and the density of immunolabeling. This correlation holds over a wide range of experimental material and conditions (e.g.,
The immunoprobe FluoroNanogold (FNG) consists of a 1.4-nm gold particle to which an affinity-purified Fab' fragment and a fluorochrome are conjugated. Thus, this ultrasmall immunoprobe combines the attributes of fluorescence and particulate detection systems. In a previous study we showed that 1.4-nm immunogold particles (in that case lacking the fluorochrome) penetrated into ultrathin cryosections to a greater extent than did colloidal gold particles (i.e., 1.4-nm > 5-nm > 10-nm) and that this led to enhanced immunolabeling at the ultrastructural level (
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Materials and Methods |
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Reagents
Goat anti-mouse FNG and goat anti-mouse 5-nm and 10-nm colloidal gold were obtained from NanoProbes (Stony Brook, NY). Goat anti-mouse 5-nm colloidal gold was also obtained from Goldmark Biologicals (Phillipsburg, NJ). Fluorescein-labeled goat anti-mouse IgG was purchased from Cappel (Durham, NC). Murine monoclonal anti-- and anti-ß-tubulin were obtained from Amersham (Arlington Heights, IL). Normal goat serum was purchased from Jackson ImmunoResearch (West Grove, PA). Gum arabic, 2-[N-morpholino] ethanesulfonic acid, sodium borohydride, sodium thiosulfate, DAPI, dimethylsulfoxide, Triton X-100, and N-formyl-Met-Leu-Phe (fMLP) were obtained from Sigma Chemical (St Louis, MO). Glutaraldehyde, osmium tetroxide, Mowiwol, and formvar were obtained from Polysciences (Warrington, PA). Epon 812 and silver lactate were obtained from Fluka (Ronkonkoma, NY), and n-propylgallate and p-phenylenediamine were supplied by Aldrich (Milwaukee, WI). Medium 199 was obtained from Gibco BRL (Gaithersburg, MD). Fetal calf serum was supplied by Intergen (Purchase, NY). All other reagents were at least reagent grade. Stock solutions of fMLP were prepared in dimethylsulfoxide and stored at -20C.
Preparation of Cells
Human leukocytes were collected onto round glass coverslips from finger prick blood as we have described previously (
Cell Incubation
Leukocytes on coverslips were incubated with the chemotactic tripeptide fMLP at 10-7 M for 5 min at 37C. Cells thus treated are more favorable for microscopic analysis than untreated cells. Cells were then fixed immediately in 0.7% glutaraldehyde in PBS for 15 min. The cells were subsequently detergent-extracted in Triton X-100 and SDS and then processed for immunocytochemical localization of MTs as we have described (
Immunocytochemistry
Coverslips were incubated in sodium borohydride (1 mg/ml) in Tris-buffered saline to block free aldehydes (two changes for 15 min each at 22C) and 5% normal goat serum in PBS to block nonspecific protein binding sites (1 hr at 22C). Subsequently, cells were incubated in a mixture of monoclonal anti-- and anti-ß-tubulin as we have described (
Silver Enhancement of FluoroNanogold and Colloidal Gold
Optimal visualization of 1.4-nm gold at both the optical and electron microscopic levels requires silver enhancement of the gold particles. FNG-labeled samples were incubated for silver enhancement of the gold particles for various times (30 sec to 15 min) using the procedure developed by Burry and associates. The silver enhancement process was stopped with a neutral pH fixer solution (for review see
Optical Microscopy
FNG-labeled MTs and FITC-IgG-labeled MTs were visualized by conventional epifluorescence microscopy. Silver- enhanced FNG-labeled and 5-nm and 10-nm colloidal gold-labeled samples were visualized by brightfield, phase-contrast, differential interference contrast (DIC), and epipolarization optics. A Nikon Optiphot microscope equipped with a Nikon B-2A filter set for fluorescence detection of FITC and a Nikon IGS filter set for epipolarization detection were employed for these studies. Nikon x100 phase-contrast, NA 1.4, and x100 DIC, NA 1.25 objective lenses were used. Micrographs were recorded on Kodak T-Max 400 film.
Confocal Microscopy
The samples were also examined by confocal microscopy using a BioRad MRC 600 operated in the reflectance mode. Images were collected after Kalman filtering and stored on an optical disk. The micrographs were printed to a GCC Technologies film recorder onto Kodak T-Max 100 film without additional manipulation of the images.
Electron Microscopy
Silver-enhanced FNG-labeled MTs were also examined by electron microscopy. After labeling with FNG and washing in the same manner as for optical microscopy, the cells were refixed in 2% glutaraldehyde in PBS for 15 minutes. The samples were then subjected to the silver enhancement procedure that was used for light microscopy for various times, from 30 sec to 5 min. After fixation of the developed silver particles and subsequent washing, the cells were incubated in 0.1% OsO4 (
In additional experiments, 5-nm colloidal gold (goat anti-mouse IgG) was used as the secondary antibody for ultra-stuctural detection of MTs. The colloidal gold was diluted either 1:15 or 1:30 (A520 was 0.103 and 0.053, respectively) over that supplied by the manufacturer in PBS-goat serum as described for FNG and FITC-IgG. After a 2-hr incubation at 22C, the unbound 5-nm gold was removed by several washes in PBS. The samples were then refixed in 2% glutaraldehyde in PBS for 15 min and then washed several times in PBS. Silver enhancement of the 5-nm colloidal gold was not employed in the EM preparations. The samples were postfixed in 0.1% OsO4 for 15 min and then washed in PBS.
In all cases, the cells on glass coverslips were dehydrated and embedded in Epon as described previously (
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Results |
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Immunocytochemical labeling of leukocyte MTs was readily achieved with FNG and conventional FITC- labeled secondary antibody using the preparation method we recently described (
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The effect of silver enhancement of the 1.4-nm FNG particles was first observed by DIC optics. At the earliest times in the silver enhancement process, MTs were detected by DIC before their optimal visualization by brightfield microscopy (Figure 3).
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Silver-enhanced 1.4-nm FNG particles were also visualized by confocal microscopy using the reflectance mode of operation (Figure 4) as well as the fluorescence mode (not shown). The reflectance imaging modality was particularly useful for defining regions of close association of cells with the substratum. These peripheral regions of close association with the substratum lack MTs.
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Silver-enhanced 1.4-nm FNG particles were readily detected in thin section preparations by TEM (Figure 5). The silver-enhanced particles were visualized in thin sections lacking counterstain and in thin sections that had been counterstained with heavy metals. The MTs were intensely labeled with the reaction product. MT labeling was evident by EM at the earliest silver enhancement time tested (i.e., 30 sec), even though MTs were not well visualized by brightfield optics in those samples. Little if any background detection of silver-enhanced gold particles was observed over the nucleus in these preparations. There were some silver-enhanced gold particles in the cytoplasm away from the MTs. However, this is probably specific labeling derived from unpolymerized tubulin that was not extracted from the cell during the preparative procedures. Microtubules were not detected in samples that did not receive silver enhancement or primary antibodies. On the other hand, labeling of MTs was absent at the EM level with 5-nm colloidal gold in these cells (data not shown). This is probably due to the preparative procedures employed in which the cells were fixed in glutaraldehyde and then subsequently extracted with detergents. The cytoplasm of cells prepared in this fashion may be sufficiently crosslinked to prevent uniform penetration of the 5-nm colloidal gold-IgG particles into the cells (see below).
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The poor labeling of leukocyte MTs achieved with the 5-nm immunogold probes at the ultrastructural level led us to a comparison of the degree of penetration of FNG to that of 5-nm colloidal gold into leukocytes. In these experiments, cells were fixed, permeabilized, exposed to primary and secondary antibodies, and incubated for silver enhancement in exactly the same manner. The only difference was in the secondary immunoprobe employed. All of the secondary antibodies were used before the expiration dates given by the manufacturers. MTs were heavily labeled after silver enhancement when FNG was used as the secondary antibody. On the other hand, when 5-nm colloidal gold served as the secondary antibody there was little if any labeling of MTs (Figure 6). As previously mentioned in conjunction with the EM results, the poor labeling of MTs achieved with 5-nm immunogold was most likely due to exclusion of the 5-nm immunoprobes from the glutaraldehyde crosslinked cytoplasm. Although there were occasional cells in these preparations (<0.5%) that showed some MT staining with 5-nm gold, they were probably damaged in some way during processing, thus allowing the colloidal gold into the cells. However, the FNG readily penetrated into the cytoplasm of leukocytes prepared in this manner, as evidenced by the labeling of the full MT array in each cell. This was an unequivocal result because examination by light microscopy allowed the examination of large numbers of cells compared to EM observation. All FNG-labeled cells displayed extensive decoration of the MTs, whereas very few cells displayed even poor MT labeling when 5-nm gold was used.
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Because of the highly dynamic nature of the MTs in human phagocytic leukocytes (
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At optimal silver enhancement times, the MTs were visualized by brightfield optics with little if any background signal. This is a very useful attribute of this approach to immunolabeling in certain situations. For example, eosinophils contain large granules that are highly autofluorescent at certain excitation and emission wavelengths (e.g.,
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The silver enhancement step is important to the optimal use of FNG at both the optical and electron microscopic levels. Comparison of Figure 2 and Figure 3 demonstrates that the level of signal derived from the silver enhancement of FNG represents a continuum that is time dependent. It should also be recognized that at later time points (i.e., postoptimal times) the signal from silver-enhanced FNG begins to degrade until ultimately there is too much background noise to permit detection of individual MTs with any of the imaging procedures employed (Figure 9).
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Discussion |
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Immunocytochemistry comprises a powerful collection of techniques that have been applied to many questions in biology and biomedicine. Much of the progress in immunocytochemistry has been dependent on development of reporter systems for demonstration of antibody binding to cell and tissue antigens. These reporter systems include fluorochromes, enzymes, and particulate probes. In the latter case, a seminal development in immunocytochemistry was the introduction of the iron-containing protein ferritin as an electron-dense marker for electron microscopy (
Colloidal gold was initially restricted to electron microscopy in its application to immunocytochemistry. However, colloidal gold immunolabeling has been applied to light microscopic labeling of cells (e.g.,
Potentially one of the most attractive avenues available for correlative microscopy is to combine fluorescence imaging with other imaging modalities (e.g., electron microscopy). Fluorescence techniques, such as immunofluorescence, display high spatial resolution and sensitivity compared to other immunocytochemical techniques routinely employed for light microscopy. The inability to correlate routinely fluorescence and electron microscopy has been a major disadvantage. Several different approaches have been employed in an effort to correlate fluorescence microscopy with other types of microscopy. An FITC-protein A-gold complex (20-nm gold) was developed for immunocytochemical labeling (
Another approach that has been employed to correlate fluorescence microscopy and electron microscopy is fluorescence photo-oxidation of diaminobenzidine (
Direct detection of ultrasmall gold (1-nm) at the EM level requires specialized equipment not normally available to biologists. For example, Stierhof and co-workers (1992) report that the best contrast of ultra-small gold particles, which had been used to immunolabel sections of resin-embedded bacteria, was obtained with a field emission STEM operated at 200 kV and equipped with a high-angle annular darkfield detector for collecting electrons that had undergone high-angle Rutherford forward scattering. Similarly, darkfield STEM has been used for high-resolution detection of individual 1.4-nm gold probes adsorbed to coated EM grids and for detection of anti-ferritin Fab' 1.4-nm gold probes labeling individual ferritin molecules (
6.3) than other silver enhancement procedures currently available. This has the advantage of not being as perturbing to cell ultrastructure.
We show that FNG is a very useful immunoprobe. It can be detected by fluorescence microscopy and by other modes of optical and electron microscopy. For example, the labeling efficiency of samples can be examined using fluorescence microscopy before silver enhancement of the FGN for electron microscopy. This provides a means to ensure proper immunoprobe localization before completing the more extensive procedures involved in preparing the sample for electron microscopy. Retention of the fluorescence signal from FNG, even after brief periods of silver enhancement, is a highly desirable feature and will permit the combination of fluorescence and other forms of optical microscopy in the same samples after immunocytochemical labeling. As further support for this contention, we were able to combine silver enhancement of FNG for detection of MTs with transmitted light and DAPI staining for fluorescence detection of nuclei and chromosomes.
When the silver enhancement schedule was optimal there was essentially no background staining. However, it must be recognized that prolonged silver enhancement causes degradation of the immunocytochemical signal. Therefore, as a practical matter, one must determine the optimal time course for silver enhancement for different types of experimental material to achieve useful immunocytochemical localization results.
Localization of MTs with conventional immunofluorescence techniques provides excellent spatial resolution of these cytoskeletal elements. The use of FNG as a secondary antibody for detection of MTs by fluorescence microscopy or by other forms of optical microscopy after silver enhancement yields high-resolution detection of these structures equal to that obtained with conventional fluorochrome-labeled IgG probes. In certain situations, (such as with eosinophils, in which background fluorescence associated with intracellular granules is a major problem), results obtained with silver-enhanced FNG may be superior to those derived from immunofluorescence.
Labeling of MTs with colloidal gold has been achieved in studies of several cell types. However, efficient labeling of these MTs with colloidal gold-secondary immunoprobes typically requires that the cells be permeabilized with detergent before stabilization of the sample by chemical fixation (e.g., 1-nm) penetrates into cells fixed before permeabilization (
The ability of FNG to penetrate into fixed cells far better than colloidal gold particles, along with the fact that this probe can be viewed directly by fluorescence microscopy or by other modes of microscopy after silver enhancement, makes it an ideal tool for correlative microscopy. The ability to resolve very small structures (e.g., MTs) equally well by fluorescence microscopy and by other types of microscopy after appropriate silver enhancement underlines the utility of FNG as an immunoprobe with great versatility.
In summary, we show FNG to be a versatile new immunoprobe. The utility of FNG primarily relates to its ability to be visualized directly by fluorescence microscopy and after silver enhancement by other modes of optical microscopy and electron microscopy. The ability of FNG to penetrate into aldehyde-fixed cells to a greater extent than colloidal gold particles also increases its versatility. FluoroNanogold should prove useful and find wide application in studies in which correlative microscopy is important.
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Acknowledgments |
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Supported in part by the Ohio State University Comprehensive Cancer Center and by NIH Grant NS 31777.
We thank Jonathan Mathias for excellent technical assistance during the course of this study. We are grateful to Dr James Hainfeld for providing samples of FluoroNanogold to test and to Dr Richard Burry for sharing reagents and his technical expertise.
Note Added in Proof
Details of the chemical properties of FNG will be presented in a separate publication by Richard Powell, James Hainfeld, and their associates.
Received for publication October 14, 1996; accepted January 13, 1997.
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