Research Interests: Biophysics, Materials Science, Microscopy, Fluorescence, Fluorescence Microscopy, and 10 moreTransmission/Scanning Electron Microscopy, Scanning Transmission Electron Microscopy, Medicine, Multidisciplinary, Quantum Dots, Cercopithecus aethiops, Animals, Quantum Dot, Eukaryotic Cells, and Microfluidic Analytical Techniques
Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic... more
Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular hetero...
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Figure S1. representative images of histology and HER2 immunohistochemistry. Figure S2. exemplary TEM images recorded from the sample 1. (PDF 8490 kb)
The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of... more
The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating...
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The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3,... more
The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individ...
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Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold... more
Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold (AuNPs) and iron oxide nanoparticles (FeO x NPs) either alone or combined. Environmental scanning electron microscopy revealed that single NPs of both types bound within minutes on the cell surface but with a distinctive difference between FeO x NPs and AuNPs. Uptake analysis studies based on laser scanning microscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry revealed intracellular appearance of both NP types in all exposure scenarios and a time-dependent increase. This increase was higher for both AuNPs and FeO x NPs during co-exposure. Cells treated with endocytotic inhibitors recovered after co-exposure, which additionally hinted that two uptake mechanisms are involved. Cross-talk between upta...
Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently... more
Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, includi...
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The development of drug resistance in cancer poses a major clinical problem. An example is human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer often treated with anti-HER2 antibody therapies, such as trastuzumab.... more
The development of drug resistance in cancer poses a major clinical problem. An example is human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer often treated with anti-HER2 antibody therapies, such as trastuzumab. Because drug resistance is rooted mainly in tumor cell heterogeneity, we examined the drug effect in different subpopulations of SKBR3 breast cancer cells and compared the results with those of a drug-resistant cell line, HCC1954. Correlative light microscopy and liquid-phase scanning transmission electron microscopy were used to quantitatively analyze HER2 responses upon drug binding, whereby many tens of whole cells were imaged. Trastuzumab was found to selectively cross-link and down-regulate HER2 homodimers from the plasma membranes of bulk cancer cells. In contrast, HER2 resided mainly as monomers in rare subpopulations of resting and cancer stem cells (CSCs), and these monomers were not internalized after drug binding. The HER2 distribution wa...
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Electron microscopy of biological cells in liquid provides unique nanoscale information. A highly attractive idea is the capability to also study physiological processes of live cells with electron microscopy. However, this idea seems... more
Electron microscopy of biological cells in liquid provides unique nanoscale information. A highly attractive idea is the capability to also study physiological processes of live cells with electron microscopy. However, this idea seems unrealistic because the minimal needed electron dose to obtain contrast is already many orders of magnitude above the lethal dose known to cause reproductive-cell death. We show here that claims of electron microscopy of viable cells in recent reports are based on a questionable interpretation of the used fluorescence live/dead assay. A practical alternative to study biological processes is correlative light and electron microscopy.
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ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental... more
ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the ...
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Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell... more
Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations...
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Description/Abstract Scanning transmission electron microscopy can be used to image specimens in water layers of several micrometers thickness, sufficiently thick to contain eukaryotic cells. We determined the resolution obtained on gold... more
Description/Abstract Scanning transmission electron microscopy can be used to image specimens in water layers of several micrometers thickness, sufficiently thick to contain eukaryotic cells. We determined the resolution obtained on gold nanoparticles above and ...
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The present study examined the prodynorphin and κ opioid receptor mRNA expression levels in the anterior cingulate and dorsolateral prefrontal cortices of subjects diagnosed with schizophrenia, bipolar disorder, or major depression as... more
The present study examined the prodynorphin and κ opioid receptor mRNA expression levels in the anterior cingulate and dorsolateral prefrontal cortices of subjects diagnosed with schizophrenia, bipolar disorder, or major depression as compared with normal controls without a psychiatric diagnosis. Multivariate analyses failed to reveal any differences in the mRNA expression levels between the four diagnostic groups, though a group
Research Interests: Cognitive Science, Depression, Schizophrenia, Bipolar Disorder, Anterior Cingulate, and 11 moreGene expression, Prefrontal Cortex, In Situ Hybridization, mRna expression levels, Major Depression, Drugs of Abuse, Opioid Receptor, Neurosciences, Affective Disorder, Cause of death, and multivariate analyses
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The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the... more
The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allow...
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A challenging aspect in understanding of biomineralization is the uncovering of the molecular mechanisms leading to self-organization of biological interfaces. Of special interest are certain intracellular processes responsible for... more
A challenging aspect in understanding of biomineralization is the uncovering of the molecular mechanisms leading to self-organization of biological interfaces. Of special interest are certain intracellular processes responsible for guiding the extracellular organization of biomaterials, a subject generally related the organization of proteins on the cell surface [1]. Transmembrane proteins connect intracellular processes with extracellular biomaterials. Sialic-acid-binding immunoglobulin-like lectins (Siglecs), for example, mediate different cellular functions via binding to extracellular sialic acids. They are involved in a number of crucial mechanisms such as cell-cell interaction signaling, and endocytosis. In mammals, there are 14 different Siglecs providing an array of different functions dependent on the length of the extracellular domains and the type of sialic acid they are recognizing. Studying the localization of Siglec proteins in the plasma membrane may lead to a better ...
Liquid scanning transmission electron microscopy (liquid STEM) was used to image individual quantum dots bound to receptors in COS7 cells. The same regions on intact fixed cells were imaged both with fluorescence microscopy and liquid... more
Liquid scanning transmission electron microscopy (liquid STEM) was used to image individual quantum dots bound to receptors in COS7 cells. The same regions on intact fixed cells were imaged both with fluorescence microscopy and liquid STEM. The obtained spatial resolution on was 3 nm. The resolution was sufficient to discriminate between quantum dots and gold nanoparticles for double labeling applications.
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P19 embryonal carcinoma cells are pluripotential and able to differentiate into a variety of cell types, including neurons, glia and fibroblast-like cells, upon retinoic acid treatment and cellular aggregation. Using reverse... more
P19 embryonal carcinoma cells are pluripotential and able to differentiate into a variety of cell types, including neurons, glia and fibroblast-like cells, upon retinoic acid treatment and cellular aggregation. Using reverse transcription-polymerase chain reaction, ligand binding and immunocytochemical methods, kappa- and delta-opioid receptors were detected in undifferentiated P19 cells. The mu-opioid receptor was not observed until one day after plating, following one essential step of differentiation, but increased in number in the four days after plating. Several different expression patterns were detected in these differentiated cells. Some cells exhibited mu- and delta-opioid receptors co-expressed, with or without K-opioid receptor; whereas some of the cells expressed only K-opioid receptor. All three opioid receptors are detected on aggregated cells which are postmitotic and also expressing neurofilaments, indicating neuronal characteristics. Furthermore, those cells express...
Research Interests: Neuroscience, Motor Control, Immunohistochemistry, Gene expression, Corticotropin Releasing Hormone, and 15 moreCerebral Cortex, Mice, Animals, Pain Perception, Human Brain, Dentate Gyrus, Periaqueductal Gray, Globus Pallidus, Gonadotropin Releasing Hormone, Locus coeruleus, Nucleus Basalis, Opioid Receptor, Neurosciences, Binding Site, and Expression pattern
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... more
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.
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Research Interests: Condensed Matter Physics, Cell, TEM, STEM, ESEM, and 3 moreResolution, Optical physics, and Nano Particle
Correlative fluorescence microscopy combined with 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... more
Correlative fluorescence microscopy combined with 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 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, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment.
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Research Interests: Electron Microscopy, Scanning Electron Microscopy, Silicon, Nanotechnology, Multidisciplinary, and 15 moreSingle molecule, Metal Nanoparticles, Cercopithecus aethiops, Gold, Animals, Fibroblast Growth Factor, Epidermal Growth Factor, Buffers, Growth Factor, Protein Binding, Dwell Time, EGF receptor, Equipment Design, Electron Microscope, and fibroblasts
Research Interests: Engineering, Chemistry, Electron Microscopy, Fluorescence Microscopy, Biology, and 15 moreNanotechnology, Medicine, Multidisciplinary, Molecular Imaging, Cercopithecus aethiops, Gold nanoparticle, Gold, Escherichia coli, Animals, Epidermal Growth Factor, Biological systems, Particle Size, Membrane Protein, Growth Factor, and Electron Microscope
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Research Interests: Electrophysiology, Long Term Potentiation, Neural Network, Multidisciplinary, Low Frequency, and 10 moreNature, High Frequency, Hippocampus, Neuronal Plasticity, Long Term Depression, SYNAPSES, Action Potentials, Psychology and Cognitive Sciences, Medical and Health Sciences, and In Vitro Techniques
We report the effective and site-specific binding of long double stranded (ds)DNA to high aspect ratio carbon nanofiber arrays. The carbon nanofibers were first coated with a thin gold layer to provide anchorage for two controllable... more
We report the effective and site-specific binding of long double stranded (ds)DNA to high aspect ratio carbon nanofiber arrays. The carbon nanofibers were first coated with a thin gold layer to provide anchorage for two controllable binding methods. One method was based on the direct binding of thiol end-labeled dsDNA. The second and enhanced method used amine end-labeled dsDNA bound with crosslinkers to a carboxyl-terminated self-assembled monolayer. The bound dsDNA was first visualized with a fluorescent, dsDNA-intercalating dye. The specific binding onto the carbon nanofiber was verified by a high resolution detection method using scanning electron microscopy in combination with the binding of neutravidin-coated fluorescent microspheres to the immobilized and biotinylated dsDNA. Functional activity of thiol end-labeled dsDNA on gold-coated nanofiber arrays was verified with a transcriptional assay, whereby Chinese hamster lung cells (V79) were impaled upon the DNA-modified nanofibers and scored for transgene expression of the tethered template. Thiol end-labeled dsDNA demonstrated significantly higher expression levels than nanofibers prepared with control dsDNA that lacked a gold-binding end-label. Employing these site-specific and robust techniques of immobilization of dsDNA onto nanodevices can be of advantage for the study of DNA/protein interactions and for gene delivery applications.
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An ElectroScan ESEM was used for in situ corrosion studies on cold drawn electropolished 316L stainless steel tube surfaces in the as-received and passivated conditions. Corrosion product was removed as it formed and the tube surface was... more
An ElectroScan ESEM was used for in situ corrosion studies on cold drawn electropolished 316L stainless steel tube surfaces in the as-received and passivated conditions. Corrosion product was removed as it formed and the tube surface was viewed before, during, and after corrosive attack. The corrosion process was followed in situ, and the sample features most susceptible to corrosion (draw lines, inclusions, etc.) were identified. In addition, X-ray photoelectron spectroscopy (XPS) was used to study the changes in surface chemistry after corrosive attack. This information provided clear evidence of relevant corrosion mechanisms and relative corrosion susceptibility.
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The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary... more
The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h...
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Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009
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Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
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Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
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Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
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Silicon microchips with thin, electron transparent silicon nitride windows provide a sample support that accommodates both light-, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation... more
Silicon microchips with thin, electron transparent silicon nitride windows provide a sample support that accommodates both light-, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and there is no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare the microchips for biological samples, and examples of images obtained using different light and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light and electron microscopy.