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The following paper appeared in Microsc. Microanal., 13, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2006); Marko, M.; Scott, J.-H.; Vicenzi, E.; DeKanich, S.; Frafjord, J.; Kotula, P.; McKernan, S., and Shields, J. (Eds.),; Cambridge University Press, New York, NY, 2007, p. 244CD.

Correlative Enzymatic and Gold Probes for Light and Electron Microscopy


R. Powell,* V. Joshi,* P. Takvorian,** A. Cali,** and J. Hainfeld*

* Nanoprobes, Incorporated, 95 Horse Block Road, Yaphank, NY, 11980
** Rutgers University, Biological Sciences, 195 University Avenue, Newark, NJ 07102


Full understanding of the structure and function of a biological system requires a variety of structural information at different levels of resolution. Simpler labeling methods, in which a single probe can provide more than one complementary data set, are highly desirable. FluoroNanogold combined fluorescent and 1.4 nm gold-labeled probes have been used for correlative fluorescent and gold labeling [1]. However, the larger gold labels often required for electron microscopy (EM) cannot be used in combined fluorescent gold probes because they quench the fluorescence through resonance energy transfer [1,2]. To address this issue, we prepared dual-labeled probes comprising horseradish peroxidase-labeled antibodies or streptavidin conjugated to 1.4 nm Nanogold, 5 nm or 10 nm colloidal gold. These may be used with fluorescent tyramide HRP substrates for fluorescent labeling, or conventional chromogenic substrates for brightfield labeling. The gold particles are visualized directly by electron microscopy, or developed using metallographic procedures such as silver enhancement or enzyme metallography [3] for light microscopy (LM) and EM visualization.

New probes were evaluated in Microsporida, parasitic organisms that are important intracellular opportunistic pathogens in AIDS and other immune compromised patients. They are responsible for chronic diarrhea, malabsorption syndromes, myositis, and disseminating infections demonstrated in all tissues of the body [4]. Microsporida form a diagnostic spore containing a coiled polar filament surrounding the sporoplasm, consisting of a nucleus or paired abutted nuclei (diplokaryon) and its associated cytoplasmic organelles. Upon activation, the polar filament everts to form a tubule through which the spore contents travel to infect a host cell [4]. Cultured RK-13 cells infected with Encephalitozoon hellem or Brachiola algerae microsporida were grown in NUNC plastic cell culture chamber slides. After immunofixing and blocking, cells were incubated with 1:100 anti- E. hellem PTP55 or anti- B. algerae PTP80 primary antibody for 30 minutes at 32°C, followed by either a 1 : 200 dilution of Nanogold and HRP-labeled IgG, or a 1 : 50 dilution of 5 or 10 nm gold and HRP-labeled IgG secondary for 30 minutes at 32°C. A 10 or 15 minute treatment with Alexa Fluor 488 tyramide revealed the polar tubes (PTs) clearly by fluorescence microscopy at similar resolution to FluoroNanogold, even using probes containing 5 or 10 nm gold, or after silver enhancement of Nanogold. EM showed specific, dense PT labeling with 5 and 10 nm gold: this is the first demonstration of correlative 5 or 10 nm gold and fluorescent labeling using a single probe. Silver enhancement produced dense labeling of PTs, observable by both LM and EM.

Enzyme metallography produced extremely intense labeling of PTs, readily observed by both LM and EM. In conjunction with fluorescent enzyme substrates, this approach enables correlative fluorescent, brightfield LM and EM studies on the same structures. With enzyme metallography, the intense labeling of the 150 nm diameter PTs allows the identification of microsporida with low power brightfield LM optics, enabling preliminary diagnosis of microsporidian infections without specialized LM optics or EM. On blots, the new probes showed significantly greater detection sensitivity than enzyme metallographic detection alone.

References

  1. R. D. Powell, C. M. R. Halsey, and J. F. Hainfeld: Microsc. Res. Tech., 42 (1998) 2.
  2. I. K. Kandela, R. Bleher, and R. M. Albrecht: Microsc. Microanal., 10 (Suppl 2) (2004) 1212CD.
  3. R. Tubbs, J. Pettay, J., et al. Appl. Immunohistochem. Mol. Morphol., 13 (2005) 371.
  4. A. Cali, L. M. Weiss, and P. M. Takvorian. J. Euk. Microbiol., 49 (2002) 164.
  5. Work was supported by NIH SBIR grant 2R44 RR022028-01 and NIH grant 5R01AI031788-15.

[Fig. 1: Combined Enzymatic and Gold Probes: Light and EM Labeling and Blots] (115k)

Figures: (a) - (h) Micrographs showing labeled polar tunes (arrows), empty spores (dark arrow-heads), and infective sporoplasm (white arrowheads) in microsporida. (a) (d) RK-13 cells infected with E. hellem microsporidia, stained using anti-PTP-55 primary and combined Nanogold and HRP-labeled secondary antibodies. (a) Fluorescence after development with Alexa Fluor 488 tyramide (63X objective); (b) brightfield after metallographic development, and (c) fluorescence after enhancement with HQ Silver then Alexa Fluor 488 tyramide (40X objective); (d) TEM after enhancement with HQ Silver. (e) (h) Labeling of polar tube proteins in B. algerae ((e) - (g)) or E. hellem (h) in cultured RK-13 cells stained using anti-PTP-80 or PTP-55 primaries respectively, and combined HRP and 5 nm gold-labeled secondary antibody: (e) fluorescence after development with Alexa Fluor 488 tyramide for 15 minutes (40X objective); (f) brightfield after development with metallographic substrate (20X objective); (g) TEM before enzyme metallographic development, showing gold particles (arrows); (h) TEM after enzyme metallography, showing intense polar tube staining. (i) Immunoblot detection of serial dilutions of biotinylated IgG using (left) HRP and Nanogoldlabeled streptavidin, and (right) HRP-streptavidin developed with enzyme metallography.


Reproduced courtesy of Cambridge University Press.

© 2005 Cambridge University Press. Used with permission.

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