TECHNICAL NOTE |
Correspondence to: Toshihiro Takizawa, Dept. of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail: ttakizawa@jichi.ac.jp
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Summary |
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Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique. (J Histochem Cytochem 48:433436, 2000)
Key Words: correlative microscopy, FluoroNanogold, immunocytochemistry, photobleaching, antifading reagents
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Introduction |
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Correlative microscopy using FluoroNanogold (FNG) and ultrathin cryosections is potentially a very useful technique for cell biological investigations. The same FNG-labeled molecules in ultrathin cryosections can be examined by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM) (
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Materials and Methods |
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Human neutrophils were isolated and prepared for ultrathin cryosectioning and immunolabeling as previously described (
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Morphometric analysis of the labeling density of FNG in electron micrographs was determined. For each of samples treated with the different mounting media for IFM, at least four neutrophils were examined. Eighteen negatives of electron micrographs were printed at the same magnification (x41,000). The areas of granule profiles were determined with NIH Image 1.62 software and the number of gold particles was determined by manual counting. A total of 71 MPO-positive granules were analyzed.
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Results |
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The immunocytochemical localization of MPO in human neutrophils, using FNG as the detection system, was initially determined by fluorescence microscopy (Fig 2A). The higher-resolution localization of MPO in the same ultrathin cryosection was then determined by EM after silver enhancement (Fig 2B). There was a one-to-one relationship between the fluorescent structures and the granule profiles labeled with silver-enhanced gold and observed by electron microscopy (Fig 2 and Fig 3). Unlabeled granule profiles were more numerous than labeled ones and represent the MPO-negative granules characteristic of these cells (e.g., the specific granules) (Fig 3). Control specimens lacking primary antibody displayed neither fluorescence nor silver-enhanced gold labeling (not shown).
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All the antifading media tested were capable of reducing the photobleaching of the FNG-associated FITC during IFM observation. However, there was a remarkable difference in the quality of the results of subsequent IEM. When DABCO was employed as an antifading reagent, silver-enhanced 1.4-nm gold particles in the azurophilic granules were dramatically diminished even when strong fluorescence signals from the same FNG probes were associated with the same structures (Fig 3D and Fig 3D'). Moreover, there was an increase in background signals in IEM (Fig 3D'). On the other hand, PPD and NPG did not adversely affect the labeling intensity of silver-enhanced FNG particles in the granules (Fig 3B, Fig 3B', 3C, and 3C'). From the quantitative point of view, the gold labeling of DABCO-treated samples was significantly lower than that of the samples without antifading reagents (p<0.001), with PPD (p<0.001), and with NPG (p<0.001) (Fig 4). In addition, DABCO itself did not cause the reduction in silver-enhanced gold unless cryosections mounted in media containing DABCO were exposed to excitation light (not shown).
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Discussion |
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In IFM, one often encounters the problem of fading of the fluorochrome due to photobleaching. One of the most convenient remedies for photobleaching of fluorescently-labeled samples in IFM is to use antifading reagents, which delay the loss of fluorescence. Compounds such as PPD, NPG, and DABCO have been widely employed for this purpose (
The possibility that prior exposure of FNG to excitation light reduces the amount of gold detected by subsequent silver enhancement has been reported (
Relatively brief excitation of FNG in the absence of antiphotobleaching agents does not appear to diminish subsequent silver enhancement of FNG. However, antiphotobleaching agents were very useful for fluorescence imaging of FNG. Nevertheless, the use of DABCO as the antiphotobleaching agent had a deleterious effect and resulted in a dramatic reduction in the amount of silver-enhanced gold. There was also an increase in the gold background staining when DABCO was used. However, the fluorescence signal in the presence of DABCO was not diminished in comparison to PPD or NPG. On the other hand, use of PPD or NPG did not lower the level of silver-enhanced gold compared to the absence of an antiphotobleaching agent.
The "DABCO effect" appears to be due to a photochemical reaction because exposure of FNG-labeled cryosections to DABCO in the absence of excitation light does not result in loss of silver-enhanced gold particles. The mechanism by which DABCO plus excitation light diminishes the specific silver-enhanced gold signal (but does not appear to alter the fluorescence signal) from FNG is unclear. However, this result is consistent with a DABCO-mediated, photochemically induced disassociation of the gold cluster compound from the parent FNG. This could explain our observation that the silver-enhanced gold background was higher when DABCO was used than when either PPD or NPG was employed to retard photobleaching. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching agent when FNG is used in correlative fluorescence and electron microscopy. In addition, if commercial antifading agents are used, they should be tested for their compatibility with the silver enhancement of FNG.
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Acknowledgments |
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Supported by grants-in-aid for Scientific Research and project grants of the Center for Molecular Medicine of Jichi Medical School from the Ministry of Education, Science, Sports, and Culture of Japan (TT) and by a grant from the American Heart Association, Ohio Valley Affiliate (JMR).
We thank Ms Kiyomi Inose, Ms Megumi Yatabe, and Ms Michiyo Soutome of Jichi Medical School for their help.
Received for publication October 11, 1999; accepted October 28, 1999.
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