329 research outputs found
Targeted Nanodiamonds for Identification of Subcellular Protein Assemblies in Mammalian Cells
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.Comment: 38 pages, 6 figures, SI section with 3 figure
Nanodiamond landmarks for subcellular multimodal optical and electron imaging.
There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable "zooming-in" to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery
- …