in-situ-tem-of-biological-assemblies-in-liquid

<em>In situ</em> TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

erratum-in-situ-tem-of-biological-assemblies-in-liquid

Erratum: <em>In situ</em> TEM of Biological Assemblies in Liquid

This corrects the article ...

Protochips, Inc.

While it has become common practice for dendrimers to deliver imaging and therapeutic agents, there are few reported examples of cellular internalization of dendrimers. Moreover, targeting of dendrimers to the mitochondria in cells has not yet been reported. Previously, we have delivered small molecule imaging agents into glioma and breast cancer cells by targeting the translocator protein (TSPO; formerly known as the peripheral benzodiazepine receptor or PBR) with a family of high-affinity conjugable ligands. The 18 kDa multimeric TSPO is expressed in steroid-producing cells, primarily on the outer mitochondrial membrane. This protein is a prime candidate for molecular targeting because tumors and other disease-related cells contain high densities of TSPO. Here, we present the synthesis, characterization, and cellular internalization into C6 rat glioma cells of a TSPO targeted dendrimer imaging agent.

TSPO targeted dendrimer imaging agent: synthesis, characterization, and cellular internalization.

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.

We present a novel microfluidic platform to examine biological assemblies at high-resolution. We have engineered a functionalized chamber that serves as a "nanoscale biosphere" to capture and maintain rotavirus double-layered particles (DLPs) in a liquid environment. The chamber can be inserted into the column of a transmission electron microscope while being completely isolated from the vacuum system. This configuration allowed us to determine the structure of biological complexes at nanometer-resolution within a self-contained vessel. Images of DLPs were used to calculate the first 3D view of macromolecules in solution. We refer to this new fluidic visualization technology as in situ molecular microscopy.

Visualizing viral assemblies in a nanoscale biosphere.

Gold nanorods are widely known for their photothermal properties to treat solid tumors. Our work demonstrates the unrealized capacity to image these reagents in liquid at high resolution using Transmission Electron Microscopy (TEM). Here we perform the first atomic measurements of functionalized nanorods in solution while visualizing their dynamic behaviour with TEM.

Visualizing nanoparticle mobility in liquid at atomic resolution.

Understanding the fundamental properties of macromolecules has enhanced the development of emerging technologies used to improve biomedical research. Currently, there is a critical need for innovative platforms that can illuminate the function of biomedical reagents in a native environment. To address this need, we have developed an in situ approach to visualize the dynamic behavior of biomedically relevant macromolecules at the nanoscale. Newly designed silicon nitride devices containing integrated "microwells" were used to enclose active macromolecular specimens in liquid for transmission electron microscopy imaging purposes. We were able to successfully examine novel magnetic resonance imaging contrast reagents, micelle suspensions, liposome carrier vehicles, and transcribing viral assemblies. With each specimen tested, the integrated microwells adequately maintained macromolecules in discrete local environments while enabling thin liquid layers to be produced.

Improved Microchip Design and Application for In Situ Transmission Electron Microscopy of Macromolecules.

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

ArticleTotal : 2	Year
Publication title Cited by 8	2013
Publication title	2024

Article	Year
TSPO targeted dendrimer imaging agent: synthesis, characterization, and cellular internalization. Bioconjugate chemistry\| PubMed ID: 19863077	2009
Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid. ACS nano\| PubMed ID: 20550177	2010
Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy. Journal of structural biology\| PubMed ID: 21440635	2011
Visualizing viral assemblies in a nanoscale biosphere. Lab on a chip\| PubMed ID: 23208001	2013
Visualizing nanoparticle mobility in liquid at atomic resolution. Chemical communications (Cambridge, England)\| PubMed ID: 23459782	2013
Improved Microchip Design and Application for In Situ Transmission Electron Microscopy of Macromolecules. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada\| PubMed ID: 24331164	2013
Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24357378	2014

Madeline J. Dukes

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