Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography
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
References (21)
- et al.
Toward fully automated high-resolution electron tomography
J. Struct. Biol.
(1996) How hearing happens
Neuron
(1997)- et al.
Automated microscopy for electron tomography
Ultramicroscopy
(1992) - et al.
Autotuning of a TEM using minimum dose
Ultramicroscopy
(1989) - et al.
An autotuning method for a TEM
Ultramicroscopy
(1987) - et al.
Computer visualization of three-dimensional image data using IMOD
J. Struct. Biol.
(1996) - et al.
Three-dimensional localization of immunogold markers using two tilted electron microscope recordings
Biophys. J.
(1995) - et al.
An auto-tuning method for focusing and astigmatism correction in HAADF-STEM, based on the image contrast transfer function
J. Electron Microsc.
(2001) - et al.
Applications of medium voltage STEM for the 3-D study of organelles within very thick sections
J. Microsc.
(1993) Histochemical demonstration of heavy metals. A revised version of the silver method suitable for both light and electron microscopy
Histochemistry
(1981)