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Abstract
Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.
Highlights
Over the past several decades, transmission electron microscopy (TEM) has allowed imaging of biological samples at the single protein level
To assess the effectiveness of antibody-conjugated NDs as labels in a cellular landscape, we have evaluated the spatial pattern of NDs twelve (12) hours after transfection into living cells relative to that of a specific, independently identifiable macromolecular assembly, the nuclear pore complex (NPC)
A large number of nuclear pores are identified in thin sections at magnifications as low as 3400x and appear as clear gaps in the nuclear envelope staining or as visible structures matching the well-defined dimensions of the NPC
Summary
Over the past several decades, transmission electron microscopy (TEM) has allowed imaging of biological samples at the single protein level. With recent advances in electron tomography, it is possible to resolve protein structures in cell sections with sub-nanometer resolution and field of view greater than 1 μm2 [1]. Attempts to investigate the structures of proteins in situ are hindered by the lack of suitable live cell-compatible labels to identify the protein being observed in TEM. We show that nanodiamonds (NDs) can be used as targeted intensity contrast labels in biological TEM to image subcellular protein assemblies inside mammalian cells. Nanodiamonds were selected as TEM labels because of their wide range of favorable physical properties for biological applications. NDs containing negatively-charged nitrogen vacancy (NV−) defect centers yield strongly fluorescent labels [2]
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