Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography

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Abstract

The localization of scarce antigens in thin sections of biological material can be accomplished by pre-embedment labeling with ultrasmall immuno-gold labels. Moreover, with this method, labeling is not restricted to the section surface but occurs throughout the section volume. Thus, when combined with electron tomography, antigens can be localized in three dimensions in relation to the 3D (three-dimensional) ultrastructure of the cell. However, for visualization in a transmission electron microscope, these labels need to be enlarged by silver or gold enhancement. The increase in particle size reduces the resolution of the antigen detection and the large particles obscure ultrastructural details in the tomogram. In this paper we show for the first time that these problems can be avoided and that ultrasmall gold labels can be localized in three dimensions without the need for gold or silver enhancement by using HAADF-STEM (high angular annular dark-field–scanning transmission electron microscopy) tomography. This method allowed us to three-dimensionally localize Aurion ultrasmall goat anti-rabbit immuno-gold labels on sections of Epon-embedded, osmium–uranium–lead-stained biological material. Calculations show that a 3D reconstruction obtained from HAADF-STEM projection images can be spatially aligned to one obtained from transmission electron microscopy (TEM) projections with subpixel accuracy. We conclude that it is possible to combine the high-fidelity structural information of TEM tomograms with the ultrasmall label localization ability of HAADF-STEM tomograms.

Introduction

Postembedment immuno-gold labeling has been shown to be very successful in localizing molecules in thin sections of biological material. If the structure under study is unaffected by the preparation, cryo-ultramicrotomy after mild chemical fixation according to Tokuyasu (1973) is the preferred method. An alternative method is freeze substitution of specimens frozen by high-pressure freezing, which is in principle a better method for structural preservation, in combination with immunolabeling (for a review see Verkleij et al., 1999). As long as one keeps in mind that structural damage due to permeabilization of the cells might be a problem, pre-embedment labeling, where antigens can be reached throughout the specimen, can be regarded as the only method for the detection and visualization of scarce antigens and three-dimensional (3D) studies at the electron microscopic level (Humbel et al., 1998). Usage of the small antibody fragment Fab in combination with ultrasmall gold particles has been shown to be successful (reviewed in Hainfeld, 1990). As these ultrasmall gold particles are not visible in Transmission electron microscopy (TEM) images of thin sections, they are subsequently enlarged by silver (Danscher, 1981) or gold (Hainfeld and Powell, 2000) enhancement. By TEM stereomicroscopy (Starink et al., 1995) the labeled antigens can be visualized in three dimensions.

As it is known that ultrasmall labels can be detected without silver enhancement in two-dimensional high angular annular dark-field–scanning transmission electron microscopy (HAADF-STEM) imaging (Hainfeld et al., 1999, Stierhoff et al., 1992), we have extended HAADF-STEM imaging to three dimensions via HAADF-STEM tomography. Successful experiments using STEM tomography of biological sections have been carried out previously (Beorchia et al., 1993) and recent experiments on HAADF-STEM tomography indicate that resolutions of at least 1 nm can be obtained for material science samples (Midgley et al., 2001; and our own experiments).

As a first step in the study, we have applied ultrasmall colloidal gold labels to the surface of conventionally prepared, Epon-embedded, osmium–uranium–lead-stained sections of bullfrog saccular hair cell sterecilia. It was thus possible to investigate the detectability of ultrasmall labels without the need for silver enhancement in the presence of heavy metal stain by the proposed method of HAADF-STEM tomography.

As the HAADF-STEM signal is approximately proportional to the square of the atomic number, the method is suited to detect small clusters of material with a higher density than the surrounding area. However, for the imaging conditions chosen, the HAADF-STEM reconstruction of the stained bullfrog stereocilia did not exhibit the same details for the biological features as a tomogram obtained from a TEM tilt series. We have therefore spatially aligned the 3D reconstruction calculated from HAADF-STEM projection images with one that was obtained from TEM projections of the same sample area, combining the advantages of both imaging modes.

Section snippets

Materials and methods

Sections of a bullfrog saccular hair cell were provided by Manfred Auer and A. James Hudspeth as part of an ongoing collaboration on the structure of the mechano-electrical transduction and adaptation machinery in stereocilia and were obtained by conventional processing of epithelial tissue including chemical fixation, a progressive lowering of temperature dehydration scheme, followed by Epon embedding. The sample was exposed to stains containing osmium, uranium, and lead. After TEM and before

Results

Figs. 1a and b show the 0° TEM and STEM projection images of the same area of an Epon-embedded section of bullfrog saccular hair cell stereocilia. Stereocilia are investigated to gain insights into the mechano-electrical transduction pathway in hearing (reviewed in Hudspeth, 1997). They are cylindrical, actin-filled rods forming a hexagonal array, protruding from the apical cellular surface of hair cells. The upper part of the images shows part of the tallest stereocilium, and the lower part

Discussion

We conclude that the small structures that were revealed by 3D reconstruction from HAADF-STEM projection images represent the ultrasmall immuno-gold labels that were absorbed to the sections, because (1) they are present at their expected location at the surfaces of the section, (2) their mass density is significantly higher than that in other parts of the reconstruction, and (3) the intensity in HAADF-STEM projection images is proportional to the square of the atomic number. Our findings are

Acknowledgements

We thank Manfred Auer (Skirball Institute of Biomolecular Medicine, NYU and The Rockefeller University) and Jim Hudspeth (HHMI and The Rockefeller University) for sections of bullfrog saccular hair cell stereocilia, Matthew Weyland and Paul Midgley (Cambridge University, UK) for information on HAADF-STEM tomography, David Mastronarde (University of Colorado at Boulder) for explanations and modifications of the IMOD software, and Koert Burger (Utrecht University, The Netherlands) for critically

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