Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography.
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.
Ziese
Ulrike
U
Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands.
Kübel
Christian
C
Verkleij
Arie
A
Koster
Abraham J
AJ
D000818:Animals; D022082:Cellular Structures / Q000648:ultrastructure; D002923:Cilia / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007150:Immunohistochemistry; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D036103:Nanotechnology / Q000379:methods; D011892:Rana catesbeiana; D014057:Tomography, X-Ray Computed / Q000379:methods*