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1.  Atomic Force Microscopic Detection Enabling Multiplexed Low-Cycle-Number Quantitative Polymerase Chain Reaction for Biomarker Assays 
Analytical Chemistry  2014;86(13):6180-6183.
Quantitative polymerase chain reaction is the current “golden standard” for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.
PMCID: PMC4082389  PMID: 24918650
2.  Nanostructured Self-Assembly of Inverted Formin 2 (INF2) and F-Actin–INF2 Complexes Revealed by Atomic Force Microscopy 
Langmuir  2014;30(25):7533-7539.
Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.
PMCID: PMC4082382  PMID: 24915113
3.  Nanofilaments on glioblastoma exosomes revealed by peak force microscopy 
Exosomes are sub-100 nm extracellular vesicles secreted by normal and cancer cells. We present a high-resolution structure of previously unidentified nanofilaments on glioblastoma-derived exosomes, using nanoscale peak force imaging. These stiff, adhesive, trypsin- and RNAse-resistant surface nanofilaments add a new dimension to the current structural knowledge of exosome-mediated intercellular communication.
PMCID: PMC3899882  PMID: 24402921
nanoparticles; glioblastoma exosomes; nanofilaments; intercellular communication; nanotechnology
4.  Mitochondrial Ca2+ uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity 
eLife  null;4:e04801.
Tightly regulated Ca2+ homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity.
eLife digest
The heart is a large muscle that pumps blood around the body by maintaining a regular rhythm of contraction and relaxation. If the heart loses this regular rhythm it works less efficiently, which can lead to life-threatening conditions.
Regular heart rhythms are maintained by changes in the concentration of calcium ions in the cytoplasm of the heart muscle cells. These changes are synchronised so that the heart cells contract in a controlled manner. In each cell, a contraction begins when calcium ions from outside the cell enter the cytoplasm by passing through a channel protein in the membrane that surrounds the cell. This triggers the release of even more calcium ions into the cytoplasm from stores within the cell. For the cells to relax, the calcium ions must then be pumped out of the cytoplasm to lower the calcium ion concentration back to the original level.
Shimizu et al. studied a zebrafish mutant—called tremblor—that has irregular heart rhythms because its heart muscle cells are unable to efficiently remove calcium ions from the cytoplasm. Embryos of the tremblor mutant were treated with a wide variety of chemical compounds with the aim of finding some that could correct the heart defect.
A compound called efsevin restores regular heart rhythms in tremblor mutants. Efsevin binds to a pump protein called VDAC2, which is found in compartments called mitochondria within the cell. Although mitochondria are best known for their role in supplying energy for the cell, they also act as internal stores for calcium. By binding to VDAC2, efsevin increases the rate at which calcium ions are pumped from the cytoplasm into the mitochondria. This restores rhythmic calcium ion cycling in the cytoplasm and enables the heart muscle cells to develop regular rhythms of contraction and relaxation. Increasing the levels of VDAC2 or another similar calcium ion pump protein in the heart cells can also restore a regular heart rhythm.
Efsevin can also correct irregular heart rhythms in human and mouse heart muscle cells, therefore the new role for mitochondria in controlling heart rhythms found by Shimizu et al. appears to be shared in other animals. The experiments have also identified the VDAC family of proteins as potential new targets for drug therapies to treat people with irregular heart rhythms.
PMCID: PMC4293673  PMID: 25588501
mitochondria; arrhythmia; calcium handling; heart; VDAC; fibrillation; human; mouse; zebrafish
5.  Green Tea Extract Selectively Targets Nanomechanics of Live Metastatic Cancer Cells 
Nanotechnology  2011;22(21):215101.
Green tea extract (GTE) is known to be a potential anticancer agent(1) with various biological activities(2, 3) yet the precise mechanism of action is still unclear. The biomechanical response of GTE treated cells taken directly from patient’s body samples was measured using atomic force microscopy(AFM)(4). We found significant increase in stiffness of GTE treated metastatic tumor cells, with a resulting value similar to untreated normal mesothelial cells, whereas mesothelial cell stiffness after GTE treatment is unchanged. Immunofluorescence analysis showed an increase in cytoskeletal-F-actin in GTE treated tumor cells, suggesting GTE treated tumor cells display mechanical, structural and morphological features similar to normal cells, which appears to be mediated by annexin-I expression, as determined by siRNA analysis of an in vitro cell line model. Our data indicates that GTE selectively targets human metastatic cancer cells but not normal mesothelial cells, a finding that is significantly advantageous compared to conventional chemotherapy agents.
PMCID: PMC3151463  PMID: 21451222
6.  Image Analysis and Length Estimation of Biomolecules Using AFM 
There are many examples of problems in pattern analysis for which it is often possible to obtain systematic characterizations, if in addition a small number of useful features or parameters of the image are known a priori or can be estimated reasonably well. Often, the relevant features of a particular pattern analysis problem are easy to enumerate, as when statistical structures of the patterns are well understood from the knowledge of the domain. We study a problem from molecular image analysis, where such a domain-dependent understanding may be lacking to some degree and the features must be inferred via machine-learning techniques. In this paper, we propose a rigorous, fully automated technique for this problem. We are motivated by an application of atomic force microscopy (AFM) image processing needed to solve a central problem in molecular biology, aimed at obtaining the complete transcription profile of a single cell, a snapshot that shows which genes are being expressed and to what degree. Reed et al. (“Single molecule transcription profiling with AFM,” Nanotechnology, vol. 18, no. 4, 2007) showed that the transcription profiling problem reduces to making high-precision measurements of biomolecule backbone lengths, correct to within 20–25 bp (6–7.5 nm). Here, we present an image processing and length estimation pipeline using AFM that comes close to achieving these measurement tolerances. In particular, we develop a biased length estimator on trained coefficients of a simple linear regression model, biweighted by a Beaton–Tukey function, whose feature universe is constrained by James–Stein shrinkage to avoid overfitting. In terms of extensibility and addressing the model selection problem, this formulation subsumes the models we studied.
PMCID: PMC4207372  PMID: 22759526
Atomic force microscopy (AFM); Beaton–Tukey; biased estimation; biomolecule; biweight; cDNA; digital contour; DNA; image processing; length estimation; linear regression; machine learning; RNA; single molecule; supervised learning
7.  Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells 
While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.
PMCID: PMC3845966  PMID: 24311969
Cardiac Differentiation; Pluripotent Embryonic Stem Cell; Matrix elasticity; Drug-selected cardiomyocyte; Synchronization; Mechanical interferometry
8.  Correlative nanoscale imaging of actin filaments and their complexes 
Nanoscale  2013;5(13):5692-5702.
Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP–actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP–actin complexes.
PMCID: PMC4030708  PMID: 23727693
9.  Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans 
Molecular oral microbiology  2012;28(2):10.1111/omi.12014.
Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram positive bacteria, including Bacillus and Lactococcus encode functions involved in protein post-export. In this study, we constructed a PrsA-deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/ membrane protein profile as well as cell surface related phenotypes, including auto-aggregation, increased surface hydrophobicity, and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface-display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to the ones identified in Bacillus, and thus is likely involved in protein post-export.
PMCID: PMC3819222  PMID: 23241367
foldase protein PrsA; protein secretion; Streptococcus mutans
10.  Atomic Force Microscopy Reveals Drebrin Induced Remodeling of F-Actin with Subnanometer Resolution 
Nano letters  2010;11(2):825-827.
We show by high-resolution atomic force microscopy analysis that drebrin A (a major neuronal actin binding protein) induced F-actin structural and mechanical remodeling involves significant changes in helical twist and filament stiffness (+55% persistence length). These results provide evidence of a unique mechanical role of drebrin in the dendrites, contribute to current molecular-level understanding of the properties of the neuronal cytoskeleton, and reflect the role of biomechanics at the nanoscale, to modulate nanofilament-structure assemblies such as F-actin.
PMCID: PMC3670797  PMID: 21175132
F-actin remodeling; drebrin; AFM; neuron cytoskeleton; nanofilament mechanics
11.  DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides 
PLoS ONE  2012;7(12):e51905.
One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.
PMCID: PMC3530553  PMID: 23300576
12.  Quantitative Nano-structural and Single Molecule Force Spectroscopy bio-molecular analysis of human saliva derived exosomes 
Exosomes are naturally occurring nanoparticles with unique structure, surface biochemistry and mechanical characteristics. These distinct nanometer sized bio-particles are secreted from the surface of oral epithelial cells into saliva, and are of interest as oral-cancer biomarkers. We use high- resolution AFM to show single vesicle quantitative differences between exosomes derived from normal and oral cancer patient’s saliva. Compared to normal exosomes (circular; 67.4 ± 2.9 nm), our findings indicate that cancer exosomes populations are significantly increased in saliva and display irregular morphologies, increased vesicle size (98.3 ± 4.6 nm) and higher inter-vesicular aggregation. At the single vesicle level, cancer exosomes exhibit significantly (P<0.05) increased CD63 surface densities. To our knowledge, it represents the first report detecting single exosome surface protein variations. Additionally, high- resolution AFM imaging of cancer saliva samples revealed discrete multi-vesicular bodies with intra-luminal exosomes enclosed. We discuss the use of quantitative, nanoscale ultra-structural and surface bio-molecular analysis of saliva exosomes, at the single vesicle and single protein level sensitivity, as a potentially new oral cancer diagnostic.
PMCID: PMC3235036  PMID: 22017459
saliva exosomes; AFM; nano-characterization; diagnostics; oral cancer
13.  Evaluation of bacteria-induced enamel demineralization using optical profilometry 
Streptococcus mutans is considered a major causative of tooth decay due to it’s ability to rapidly metabolize carbohydrates such as sucrose. One prominent excreted end product of sucrose metabolism is lactic acid. Lactic acid causes a decrease in the pH of the oral environment with subsequent demineralization of the tooth enamel. Biologically relevant bacteria-induced enamel demineralization was studied.
Optical profiling was used to measure tooth enamel decay with vertical resolution under one nanometer and lateral features with optical resolution as a result of S. mutans biofilm exposure. Comparison measurements were made using AFM.
After 72 hr of biofilm exposure the enamel displayed an 8-fold increase in the observed roughness average, (Ra), as calculated over the entire measured array. Similarly, the average root mean square (RMS) roughness, RRMS, of the enamel before and after biofilm exposure for 3 days displayed a 7-fold increase. Further, the direct effect of chemically induced enamel demineralization using biologically relevant organic acids was shown. Optical profiles of the enamel surface after addition of a 30% lactic acid solution showed a significant alteration in the surface topography with a corresponding increase in respective surface roughness statistics. Similar measurements with 10% citric acid over seconds and minutes give insight into the demineralization process by providing quantitative measures for erosion rates: comparing surface height and roughness as metrics.
The strengths of optical profilometry as an analytical tool for understanding and analyzing biologically relevant processes such as biofilm induced tooth enamel demineralization were demonstrated.
PMCID: PMC3454478  PMID: 19732947
enamel erosion; optical profilometry; biofilm; Streptococcus mutans; enamel demineralization; citric acid; lactic acid; AFM
14.  In Situ Mechanical Interferometry of Matrigel™ Films 
Many biological materials and cell substrates are very soft (Young's modulus < 500 Pa) and it is difficult to characterize their mechanical properties. Here we report local elasticity of the surface layers of Matrigel™ films used for cell culture. We used a new measurement technology, Mechanical Imaging Interferometry, to obtain point mechanical measurements over micron-sized areas. The median values of 650 Pa +/- 400 Pa (# measurements, n=50), determined by the Hertz contact model, agree well with bulk measurements, however on the micro-scale the films were heterogeneous and contained regions distinctly stiffer than average (1-2 kPa). The first measurement of yield strengths of 170 Pa +/- 100 Pa (n=43) indicate that Matrigel™ films deform plastically at stress levels of similar scale to cell tractional forces.
PMCID: PMC3437936  PMID: 19049394
15.  Neuromorphic Atomic Switch Networks 
PLoS ONE  2012;7(8):e42772.
Efforts to emulate the formidable information processing capabilities of the brain through neuromorphic engineering have been bolstered by recent progress in the fabrication of nonlinear, nanoscale circuit elements that exhibit synapse-like operational characteristics. However, conventional fabrication techniques are unable to efficiently generate structures with the highly complex interconnectivity found in biological neuronal networks. Here we demonstrate the physical realization of a self-assembled neuromorphic device which implements basic concepts of systems neuroscience through a hardware-based platform comprised of over a billion interconnected atomic-switch inorganic synapses embedded in a complex network of silver nanowires. Observations of network activation and passive harmonic generation demonstrate a collective response to input stimulus in agreement with recent theoretical predictions. Further, emergent behaviors unique to the complex network of atomic switches and akin to brain function are observed, namely spatially distributed memory, recurrent dynamics and the activation of feedforward subnetworks. These devices display the functional characteristics required for implementing unconventional, biologically and neurally inspired computational methodologies in a synthetic experimental system.
PMCID: PMC3412809  PMID: 22880101
16.  Mechanical Interferometry Imaging for Creep Modeling of the Cornea 
Nanoindentation by magnetic microspheres imaged by optical interferometry permits determination of the viscoelastic properties of fine local regions of each layer of the cornea. This approach provides robust biomechanical data on corneal creep behavior that scales reliably with the magnitude of applied force throughout the tissue.
A novel nanoindentation technique was used to biomechanically characterize each of three main layers of the cornea by using Hertzian viscoelastic formulation of creep, the deformation resulting from sustained-force application.
The nanoindentation method known as mechanical interferometry imaging (MII) with <1-nm displacement precision was used to observe indentation of bovine corneal epithelium, endothelium, and stroma by a spherical ferrous probe in a calibrated magnetic field. For each specimen, creep testing was performed using two different forces for 200 seconds. Measurements for single force were used to build a quantitative Hertzian model that was then used to predict creep behavior for another imposed force.
For all three layers, displacement measurements were highly repeatable and were well predicted by Hertzian models. Although short- and long-term stiffnesses of the endothelium were highest of the three layers at 339.2 and 20.2 kPa, respectively, both stromal stiffnesses were lowest at 100.4 and 3.6 kPa, respectively. Stiffnesses for the epithelium were intermediate at 264.6 and 12.2 kPa, respectively.
Precise, repeatable measurements of corneal creep behavior can be conveniently obtained using MII at mechanical scale as small as one cell thickness. When interpreted in analytical context of Hertzian viscoelasticity, MII technique proved to be a powerful tool for biomechanical characterization of time-dependent biomechanics of corneal regions.
PMCID: PMC3388749  PMID: 21969299
17.  Localized Nanoscopic Surface Measurements of Nickel-Modified Mica for Single Molecule DNA Sequence Sampling 
ACS applied materials & interfaces  2010;2(11):3249-3256.
Cleaved, cation-derivatized Muscovite mica is utilized extensively in AFM imaging due to its flatness over large areas (millimeter cleavage planes with local RMS roughness <0.3 nm), ease of preparation and ability to adsorb charged biomolecules such as DNA1–3. In particular, NiCl2 treatment has become a common method for controlling DNA adsorption on mica substrates while retaining the mica’s ultra flat surface4. While several studies have modeled the mica:metal-ion:DNA system using macroscopic colloidal theory (DLVO, etc) 4–8, Ni-mica’s physicochemical properties have not been well characterized on the nanoscale. Efforts to manipulate and engineer DNA nanostructures would benefit greatly from a better understanding of the surface chemistry of Ni:mica. Here we present in-situ, nanometer- and attogram-scale measurements, and thermodynamic simulation results that show the surface chemistry of nickel treated mica is more complex than generally appreciated by AFM practitioners, due to metal ion speciation effects present at neutral pH. We also show that under certain preparations, Ni:Mica allows in situ, nanoscopic nucleotide sequence mapping within individual surface-adsorbed DNA molecules by permitting localized, controlled desorption of the double helix by soluble DNA binding enzymes. These results should aid efforts to precisely control DNA:mica binding affinity, particularly at physiological pH ranges required by enzymatic biochemistry (pH 7.0–8.5)4–8, and facilitate development of more complex and useful biochemical manipulations of adsorbed DNA, such as single molecule sequencing.
PMCID: PMC3010388  PMID: 21033675
Single Molecule DNA Sequence; Surface-Modified Mica; ToF-SIMS; AFM and surface analysis
18.  Characterization of Ocular Tissues Using Microindentation and Hertzian Viscoelastic Models 
Microindentation permits biomechanical characterization of small specimens of ocular tissues and demonstrates that although properties of periocular fatty tissues vary markedly by location, comparable bovine and human tissues behave similarly.
The authors applied a novel microindentation technique to characterize biomechanical properties of small ocular and orbital tissue specimens using the Hertzian viscoelastic formulation, which defines material viscoelasticity in terms of the contact pressure required to maintain deformation by a harder body.
They used a hard spherical indenter having 100 nm displacement and 100 μg force precision to impose small deformations on fresh bovine sclera, iris, crystalline lens, kidney fat, orbital pulley tissue, and orbital fatty tissue; normal human orbital fat, eyelid fat, and dermal fat; and orbital fat associated with thyroid eye disease. For each tissue, stress relaxation testing was performed using a range of ramp displacements. Results for single displacements were used to build quantitative Hertzian models that were, in turn, compared with behavior for other displacements. Findings in orbital tissues were correlated with quantitative histology.
Viscoelastic properties of small specimens of orbital and ocular tissues were reliably characterized over a wide range of rates and displacements by microindentation using the Hertzian formulation. Bovine and human orbital fatty tissues exhibited highly similar elastic and viscous behaviors, but all other orbital tissues exhibited a wide range of biomechanical properties. Stiffness of fatty tissues tissue depended strongly on the connective tissue content.
Relaxation testing by microindentation is a powerful method for characterization of time-dependent behaviors of a wide range of ocular and orbital tissues using small specimens, and provides data suitable to define finite element models of a wide range of tissue interactions.
PMCID: PMC3109037  PMID: 21310907
19.  Structural- mechanical characterization of nanoparticles- Exosomes in human saliva, using correlative AFM, FESEM and force spectroscopy 
ACS nano  2010;4(4):1921-1926.
All living systems contain naturally occurring nanoparticles with unique structural, biochemical and mechanical characteristics. Specifically, human saliva exosomes secreted by normal cells into saliva via exocytosis, are novel biomarkers showing tumor-antigen enrichment during oral cancer. Here we show the substructure of single human saliva exosomes, using a new ultra sensitive low force Atomic Force Microscopy (AFM) exhibiting sub-structural organization unresolvable in Electron Microscopy. We correlate the data with Field Emission Scanning Electron Microscopy (FESEM) and AFM images to interpret the nanoscale structures of exosomes under varying forces. Single exosomes reveal reversible mechanical deformation displaying distinct elastic, 70-100nm tri-lobed membrane with sub-structures carrying specific trans-membrane receptors. Further, we imaged and investigated, using force spectroscopy with antiCD63 IgG functionalized AFM tips, highly specific and sensitive detection of antigenCD63, potentially useful cancer markers on individual exosomes. The quantitative nanoscale morphological, biomechanical and surface biomolecular properties of single saliva exosomes, are critical for the applications of exosomes for cancer diagnosis and as a model for developing new cell delivery systems.
PMCID: PMC2866049  PMID: 20218655
Exosomes; saliva nanoparticles; nanocharacterization; Force spectroscopy; CD63 membrane receptors
20.  Mechanical Interferometry of Nanoscale Motion and Local Mechanical Properties of Living Zebrafish Embryos 
ACS nano  2009;3(8):2090-2094.
We present an interferometric imaging technique that permits local measurement of mechanical properties and nanomechancial motion in small living animals. Measurements of nanomechanical properties and spatially resolved pulsations of <60 nm were recorded for the developing eye of a living zerbrafish (Danio rerio) embryo, an important model organism. We also used magnetic microreflectors to conduct contact nanomechanical indentation measurements of the stiffness of the embryonic eye.
PMCID: PMC3081709  PMID: 19591446
interferometry; zerbrafish; nano-mechanical properties
21.  High throughput cell nanomechanics with mechanical imaging interferometry 
Nanotechnology  2008;19(23):235101-.
The dynamic nanomechanical properties of a large number of cells (up to hundreds), measured in parallel with high throughput, are reported. Using NIH 3T3 and HEK 293T fibroblasts and actin depolymerizing drugs, we use a novel nanotechnology to quantify the local viscoelastic properties with applied forces of 20 pN–20 nN, a spatial resolution of <20 nm, and a mechanical dynamic range of several Pa up to ~200 kPa. Our approach utilizes imaging interferometry in combination with reflective, magnetic probes attached to cells. These results indicate that mechanical imaging interferometry is a sensitive and scalable technology for measuring the nanomechanical properties of large arrays of live cells in fluid.
PMCID: PMC2925287  PMID: 20737027
22.  Single molecule transcription profiling with AFM* 
Nanotechnology  2007;18(4):44032.
Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations.
PMCID: PMC2922717  PMID: 20721301
23.  Dynamic mechanical oscillations during metamorphosis of the monarch butterfly 
The mechanical oscillation of the heart is fundamental during insect metamorphosis, but it is unclear how morphological changes affect its mechanical dynamics. Here, the micromechanical heartbeat with the monarch chrysalis (Danaus plexippus) during metamorphosis is compared with the structural changes observed through in vivo magnetic resonance imaging (MRI). We employ a novel ultra-sensitive detection approach, optical beam deflection, in order to measure the microscale motions of the pupae during the course of metamorphosis. We observed very distinct mechanical contractions occurring at regular intervals, which we ascribe to the mechanical function of the heart organ. Motion was observed to occur in approximately 15 min bursts of activity with frequencies in the 0.4–1.0 Hz range separated by periods of quiescence during the first 83 per cent of development. In the final stages, the beating was found to be uninterrupted until the adult monarch butterfly emerged. Distinct stages of development were characterized by changes in frequency, amplitude, mechanical quality factor and de/repolarization times of the mechanical pulsing. The MRI revealed that the heart organ remains functionally intact throughout metamorphosis but undergoes morphological changes that are reflected in the mechanical oscillation.
PMCID: PMC2610326  PMID: 18682363
monarch butterfly; heart; mechanics; oscillation; development; metamorphosis
24.  Nanocharacterization in Dentistry 
About 80% of US adults have some form of dental disease. There are a variety of new dental products available, ranging from implants to oral hygiene products that rely on nanoscale properties. Here, the application of AFM (Atomic Force Microscopy) and optical interferometry to a range of dentistry issues, including characterization of dental enamel, oral bacteria, biofilms and the role of surface proteins in biochemical and nanomechanical properties of bacterial adhesins, is reviewed. We also include studies of new products blocking dentine tubules to alleviate hypersensitivity; antimicrobial effects of mouthwash and characterizing nanoparticle coated dental implants. An outlook on future “nanodentistry” developments such as saliva exosomes based diagnostics, designing biocompatible, antimicrobial dental implants and personalized dental healthcare is presented.
PMCID: PMC2904930  PMID: 20640166
nano-characterization; dentistry; biofilms; bacterial adhesins; implants; dentine tubule; afm; interferometry; nanodentistry
25.  Live Cell Interferometry Reveals Cellular Dynamism During Force Propagation 
ACS nano  2008;2(5):841-846.
Cancer and many other diseases are characterized by changes in cell morphology, motion, and mechanical rigidity. However, in live cell cytology, stimulus-induced morphologic changes typically take 10–30 min to detect. Here, we employ live-cell interferometry (LCI) to visualize the rapid response of a whole cell to mechanical stimulation, on a time scale of seconds, and we detect cytoskeletal remodeling behavior within 200 s. This behavior involved small, rapid changes in cell content and miniscule changes in shape; it would be difficult to detect with conventional or phase contrast microscopy alone and is beyond the dynamic capability of AFM. We demonstrate that LCI provides a rapid, quantitative reconstruction of the cell body with no labeling. This is an advantage over traditional microscopy and flow cytometry, which require cell surface tagging and/or destructive cell fixation for labeling
PMCID: PMC2733939  PMID: 19206480
interferometry; cytoskeleton remodeling; live-cell imaging

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