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Dendritic cells (DCs) can infiltrate tight junctions of the epithelium to collect remote antigens during immune surveillance. While elongated membrane structures represent a plausible structure to perform this task, their functional mechanisms remain elusive owing to the lack of high-resolution characterizations in live DCs. Here, we developed fluorescent artificial antigens (FAAs) based on quantum dots coated with polyacrylic acid. Single-particle tracking of FAAs enables us to superresolve the membrane fiber network responsible for the antigen uptake. Using the DC2.4 cell line as a model system, we discovered the extensive membrane network approaching 200 μm in length with tunnel-like cavities about 150 nm in width. The membrane fiber network also contained heterogeneous circular migrasomes. Disconnecting the membrane network from the cell body decreased the intracellular FAA density. Our study enables mechanistic investigations of DC membrane networks and nanocarriers that target this mechanism.

Antibody-antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody-antigen interactions for SMLM-based quantitative superresolution imaging.

Summary: Metformin is the front-line treatment for type 2 diabetes worldwide. It acts via effects on glucose and lipid metabolism in metabolic tissues, leading to enhanced insulin sensitivity. Despite significant effort, the molecular basis for metformin response remains poorly understood, with a limited number of specific biochemical pathways studied to date. To broaden our understanding of hepatic metformin response, we combine phospho-protein enrichment in tissue from genetically engineered mice with a quantitative proteomics platform to enable the discovery and quantification of basophilic kinase substrates in vivo. We define proteins whose binding to 14-3-3 are acutely regulated by metformin treatment and/or loss of the serine/threonine kinase, LKB1. Inducible binding of 250 proteins following metformin treatment is observed, 44% of which proteins bind in a manner requiring LKB1. Beyond AMPK, metformin activates protein kinase D and MAPKAPK2 in an LKB1-independent manner, revealing additional kinases that may mediate aspects of metformin response. Deeper analysis uncovered substrates of AMPK in endocytosis and calcium homeostasis. : Metformin is a potential anti-aging and anti-cancer therapy and a treatment for diabetes. Stein et al. investigate metformin-induced signaling in the liver, using 14-3-3 binding to identify phosphorylation events acting as dominant regulators of target protein activity. Kinases (PKD, MK2) activated by metformin independent of LKB1/AMPK and other targets of metformin are identified. Keywords: metformin, AMPK3, diabetes, aging, STIM1, calcium, PKD1, LKB1, liver, kinases

The spatial organization of T cell receptors (TCRs) correlates with membrane-associated signal amplification, dispersion, and regulation during T cell activation. Despite its potential clinical importance, quantitative analysis of the spatial arrangement of TCRs from standard fluorescence images remains difficult. Here, we report Statistical Classification Analyses of Membrane Protein Images or SCAMPI as a technique capable of analyzing the spatial arrangement of TCRs on the plasma membrane of T cells. We leveraged medical image analysis techniques that utilize pixel-based values. We transformed grayscale pixel values from fluorescence images of TCRs into estimated model parameters of partial differential equations. The estimated model parameters enabled an accurate classification using linear discrimination techniques, including Fisher Linear Discriminant (FLD) and Logistic Regression (LR). In a proof-of-principle study, we modeled and discriminated images of fluorescently tagged TCRs from Jurkat T cells on uncoated cover glass surfaces (Null) or coated cover glass surfaces with either positively charged poly-L-lysine (PLL) or TCR cross-linking anti-CD3 antibodies (OKT3). Using 80 training images and 20 test images per class, our statistical technique achieved 85% discrimination accuracy for both OKT3 versus PLL and OKT3 versus Null conditions. The run time of image data download, model construction, and image discrimination was 21.89 s on a laptop computer, comprised of 20.43 s for image data download, 1.30 s on the FLD-SCAMPI analysis, and 0.16 s on the LR-SCAMPI analysis. SCAMPI represents an alternative approach to morphology-based qualifications for discriminating complex patterns of membrane proteins conditioned on a small sample size and fast runtime. The technique paves pathways to characterize various physiological and pathological conditions using the spatial organization of TCRs from patient T cells.

We report single-particle characterization of membrane-penetrating semiconductor quantum dots (QDs) in T cell lymphocytes. We functionalized water-soluble CdSe/CdZnS QDs with a cell-penetrating peptide composed of an Asp-Ser-Ser (DSS) repeat sequence. DSS and peptide-free control QDs displayed concentration-dependent internalization. Intensity profiles from single-particle imaging revealed a propensity of DSS-QDs to maintain a monomeric state in the T cell cytosol, whereas control QDs formed pronounced clusters. Single-particle tracking showed a direct correlation between individual QD clusters' mobility and aggregation state. A significant portion of control QDs colocalized with an endosome marker inside the T cells, while the percentage of DSS-QDs colocalized dropped to 9%. Endocytosis inhibition abrogated the internalization of control QDs, while DSS-QD internalization only mildly decreased, suggesting an alternative cell-entry mechanism. Using 3D single-particle tracking, we captured the rapid membrane-penetrating activity of a DSS-QD. The ability to characterize membrane penetrating activities in live T cells creates inroads for the optimization of gene therapy and drug delivery through the use of novel nanomaterials.

Transient receptor potential melastatin 7 (TRPM7) contributes to a variety of physiological and pathological processes in many tissues and cells. With a widespread distribution in the nervous system, TRPM7 is involved in animal behaviors and neuronal death induced by ischemia. However, the physiological role of TRPM7 in central nervous system (CNS) neuron remains unclear. Here, we identify endocytic defects in neuroendocrine cells and neurons from TRPM7 knockout (KO) mice, indicating a role of TRPM7 in synaptic vesicle endocytosis. Our experiments further pinpoint the importance of TRPM7 as an ion channel in synaptic vesicle endocytosis. Ca2+ imaging detects a defect in presynaptic Ca2+ dynamics in TRPM7 KO neuron, suggesting an importance of Ca2+ influx via TRPM7 in synaptic vesicle endocytosis. Moreover, the short-term depression is enhanced in both excitatory and inhibitory synaptic transmissions from TRPM7 KO mice. Taken together, our data suggests that Ca2+ influx via TRPM7 may be critical for short-term plasticity of synaptic strength by regulating synaptic vesicle endocytosis in neurons.

Single-molecule imaging has provided new insights on weak transient biomolecular interactions with micromolar to millimolar affinity. However, the limited duration of observation has hindered the study of strong and reversible interactions with sub-nanomolar affinity. We report single-molecule interaction microscopy (SMIM), which combines point accumulation for imaging in nanoscale topography (PAINT) with extended imaging durations that enables the study of antibody binding kinetics in the cellular environment. SMIM revealed heterogeneous binding kinetics and the effect of concentration and antibody valency on the association and dissociation rates on antibody-antigen interactions in their cellular environments. We thereby demonstrate SMIM as a versatile single-molecule technique for studying strong, transient biomolecular interactions.

The aggregation state and endosomal trapping of engineered nanocarriers once internalized into cells remain poorly characterized. Here, we visualized the membrane penetrating dynamics of semiconductor quantum dots (QDs) into the cytosol of T cells on a single-cell and single-nanoparticle basis. We water solubilized CdSe/CdZnS QDs with polymer encapsulants functionalized with a cell-penetrating peptide composed of an Asp-Ser-Ser (DSS) repeat sequence. T cells tolerated the 24-h incubation with QDs at concentrations of 5 nM or lower. Single-particle imaging demonstrated that the number of internalized nanoparticles was dependent upon the concentration of the probes for both control (peptide-free) and DSS-QDs. DSS-QDs were mostly distributed as monomers, whereas the control QDs were aggregated into clusters. Single-particle tracking using total internal reflection and highly inclined illumination showed that DSS-QDs were stationary near the activating surface and mobile within the cytosol of the T cell. A correlation exhibited between the mobility and aggregation state of individual QD clusters, with monomeric DSS-QDs showing the highest mobility. In addition, monomeric DSS-QDs displayed much faster diffusion than the endosomes. A small-molecule endosome marker confirmed the absence of colocalization between endosomes and DSS-QDs, indicating their endosomal escape. The ability to deliver and track individual QDs in the cytosol of live T cells creates inroads for the optimization of drug delivery and gene therapy through the use of nanoparticles.

We demonstrate a statistical modeling technique to recognize T cell responses to different external environmental conditions using membrane distributions of T cell receptors. We transformed fluorescence images of T cell receptors from each T cell into estimated model parameters of a partial differential equation. The model parameters enabled the construction of an accurate classification model using linear discrimination techniques. We further demonstrated that the technique successfully differentiated immobilized T cells on non-activating and activating surfaces. Compared to machine learning techniques, our statistical technique relies upon robust image-derived statistics and achieves effective classification with a limited sample size and a minimal computational footprint. The technique provides an effective strategy to quantitatively characterize the global distribution of membrane receptors under various physiological and pathological conditions.

We introduce a cost-effective imaging method using droplets of consumer-grade cooking oil and a cell phone camera. While cooking oil droplets are not specifically designed for imaging, we found that, similar to immersion oil, they were more resistant to evaporation than water, thereby enabling long-term imaging. We harnessed their close refractive index to immersion oil and demonstrated their use as lenses for cell phone microscopy. Our new method enables stable droplet-based optical imaging primarily using household materials without specialized setups or manufacturing processes.

Maturity onset diabetes of the young (MODY) is a monogenic form of diabetes attributed to a highly penetrant variant in one of several genes, including GCK. GCK-MODY rarely requires pharmacotherapy, however, the clinical management of pregnant mothers depends on whether the fetus inherited the GCK variant. The classic GCK-MODY phenotype is young (diagnosed before 25 years), lean (BMI < 25 mg/kg2), with stable, mild hyperglycemia (Hba1c 5.8-7.6%). We hypothesized that strict application of these guidelines has led to under-reporting of GCK-MODY (prevalence 1.1 per 1000). In a cohort of 92,412 individuals from the DiscovEHR study, we identified 252 heterozygous carriers of 88 rare GCK variants (minor allele frequency < 0.02%). None had an existing diagnosis of monogenic diabetes. The median BMI of GCK carriers was 35 mg/kg2, compared to 31 mg/kg2 for nondiabetic noncarriers and 38 mg/kg2 for noncarriers with type 2 diabetes (T2D). Excluding variants with no evidence of hyperglycemia in 50% of the carriers resulted in 70 variants in 213 individuals. GCK-MODY variant carriers had mean HbA1c, fasting blood glucose (FBG), and random glucose measurements that were greater than nondiabetic non-carriers but less than those with T2D. Kaplan-Meier analysis adjusted for left-truncation bias showed that the age at which 50% of GCK-MODY subjects diagnosed with impaired fasting glucose and diabetes is 35 and 55 years, respectively, compared to 60 and 65 years in non-carriers with T2D (p< 2e-16). Within-individual variability of lifetime FBG measurements of GCK-MODY carriers was less than noncarriers with T2D (GCK-MODY, 7.9 ± 2.0 mg/dL; T2D, 17.6 ± 0.03 mg/dl, p Disclosure U.L. Mirshahi: None. J.M. Goehringer: None. Y. Hu: None. A.H. Wardeh: None. J. Williams: None. C.B. Manney: None. J.C. Staples: Employee; Self; Regeneron Pharmaceuticals. J.B. Leader: None. A.R. Shuldiner: Employee; Self; Regeneron Pharmaceuticals. Stock/Shareholder; Self; Rhythm Pharmaceuticals, Inc. T.I. Pollin: Other Relationship; Self; Regeneron Genetics Center. D.J. Carey: None.

Over the past ~20 years there has been considerable effort towards the optimization of the photophysical properties of emissive nanomaterials to engender efficacy for biological imaging and sensing. The development of core-shell CdSe/ZnS and CdSe/CdS quantum dots (QDs) was instrumental due to those materials’ resistance to photobleaching and high quantum yields. However, these particles experience fluorescence intermittency (“blinking”), which is detrimental for single particle tracking studies. Furthermore, ~10 nanosecond lifetimes are not compatible with time-gating visualization, a technique that reduces background noise. Last, cytosolic delivery of QDs is a well-known problem due to the propensity for QDs to become sequestered in the endosomes of live cells. Presented are developments that address these issues. Long lifetimes, >100 ns, and non-blinking behavior have been realized in “giant” quantum dots with a highly alloyed core/shell interface. This results in type-II behavior that separates the electron and hole, which slows emissive recombination. Also presented is a novel cell-penetrating peptide that can be conjugated to surfactants that are commonly employed for QD water solubilization. These materials can be passively delivered to the cytosol of live T-cell lymphocytes. Single particle tracking studies reveal fast diffusion of single dots that are not consistent with endosomal capture, which is observed in control nanomaterials. These developments create new inroads for chemical and biological sensing studies inside of live cells for basic research and diagnostic purposes. Figure 1

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

Synergistic developments in advanced fluorescent imaging and labeling techniques enable direct visualization of the chromatin structure and dynamics at the nanoscale level and in live cells. Super-resolution imaging encompasses a class of constantly evolving techniques that break the diffraction limit of fluorescence microscopy. Structured illumination microscopy provides a twofold resolution improvement and can readily achieve live multicolor imaging using conventional fluorophores. Single-molecule localization microscopy increases the spatial resolution by approximately 10-fold at the expense of slower acquisition speed. Stimulated emission-depletion microscopy generates a roughly fivefold resolution improvement with an imaging speed proportional to the scanning area. In parallel, advanced labeling strategies have been developed to "light up" global and sequence-specific DNA regions. DNA binding dyes have been exploited to achieve high labeling densities in single-molecule localization microscopy and enhance contrast in correlated light and electron microscopy. New-generation Oligopaint utilizes bioinformatics analyses to optimize the design of fluorescence in situ hybridization probes. Through sequential and combinatorial labeling, direct characterization of the DNA domain volume and length as well as the spatial organization of distinct topologically associated domains has been reported. In live cells, locus-specific labeling has been achieved by either inserting artificial loci next to the gene of interest, such as the repressor-operator array systems, or utilizing genome editing tools, including zinc finer proteins, transcription activator-like effectors, and the clustered regularly interspaced short palindromic repeats systems. Combined with single-molecule tracking, these labeling techniques enable direct visualization of intra- and inter-chromatin interactions. This article is categorized under: Laboratory Methods and Technologies > Imaging.

Single-molecule super-resolution imaging is a new promising tool for investigation of sub-cellular structures. Concurrently, light-sheet microscopy, also known as selective plane illumination microscopy (SPIM), has gained rapid favor with the imaging community in developmental biology due to its fast speed, high contrast, deep penetration, and low phototoxicity. While nearly a dozen reviews thoroughly describe the development of light-sheet microscopy and its technological breakthroughs with a main focus on improving the 3D imaging speed of fish embryos, central nervous system, and other tissues, few have addressed the potential of combining light-sheet microscopy and localization-based super-resolution imaging to achieve sub-diffraction-limited resolution. Adapting light-sheet illumination for single-molecule imaging presents unique challenges for instrumentation and reconstruction algorithms. In this Minireview, we provide an overview of the recent developments that address these challenges. We compare different approaches in super-resolution and light-sheet imaging, address advantages and limitations in each approach, and outline future directions of this emerging field.

Photobleaching of fluorescent proteins (FPs) is a major limitation to their use in advanced microscopy, and improving photostability remains highly challenging due to limited understanding of its molecular mechanism. Here we discovered a new mechanism to increase FP photostability. Cysteine oxidation, implicated in only photobleaching before, was found to drastically enhance FP photostability to the contrary. We generated a far-red FP mStable by introducing a cysteine proximal to the chromophore. Upon illumination, this cysteine was oxidized to sulfinic and sulfonic acids, enabling mStable more photostable than its ancestor mKate2 by 12-fold and surpassing other far-red FPs. mStable outperformed in laser scanning confocal imaging and super-resolution structured illumination microscopy. Moreover, photosensitization to oxidize a cysteine similarly introduced in another FP mPlum also increased its photostability by 23-fold. This post-folding cysteine sulfoxidation cannot be simply substituted by the isosteric aspartic acid, representing a unique mechanism valuable for engineering better photostability into FPs.

When plasmonic nanoparticles (NPs) are internalized by cells and agglomerate within intracellular vesicles, their optical spectra can shift and broaden as a result of plasmonic coupling of NPs in close proximity to one another. For such optical changes to be accounted for in the design of plasmonic NPs for light-based biomedical applications, quantitative design relationships between designable factors and spectral shifts need to be established. Here we begin building such a framework by investigating how functionalization of gold NPs (AuNPs) with biocompatible poly(ethylene) glycol (PEG), and the serum conditions in which the NPs are introduced to cells impact the optical changes exhibited by NPs in a cellular context. Utilizing darkfield hyperspectral imaging, we find that PEGylation decreases the spectral shifting and spectral broadening experienced by 100 nm AuNPs following uptake by Sk-Br-3 cells, but up to a 33 ± 12 nm shift in the spectral peak wavelength can still occur. The serum protein-containing biological medium also modulates the spectral changes experienced by cell-exposed NPs through the formation of a protein corona on the surface of NPs that mediates NP interactions with cells: PEGylated AuNPs exposed to cells in serum-free conditions experience greater spectral shifts than in serum-containing environments. Moreover, increased concentrations of serum (10, 25, or 50 %) result in the formation of smaller intracellular NP clusters and correspondingly reduced spectral shifts after 5 and 10 h NP-cell exposure. However, after 24 h, NP cluster size and spectral shifts are comparable and become independent of serum concentration. By elucidating the impact of PEGylation and serum concentration on the spectral changes experienced by plasmonic NPs in cells, this study provides a foundation for the optical engineering of plasmonic NPs for use in biomedical environments. Electronic supplementary material The online version of this article (doi:10.1186/s11671-016-1524-4) contains supplementary material, which is available to authorized users.

Nanotechnology-based cancer treatment approaches potentially provide localized, targeted therapies that aim to enhance efficacy, reduce side effects, and improve patient quality of life. Gold-nanoparticle-mediated hyperthermia shows particular promise in animal studies, and early clinical testing is currently underway. In this article, the rapidly evolving field of gold nanoparticle thermal therapy is reviewed, highlighting recent literature and describing current challenges to clinical translation of the technology.

We investigate surface-enhanced Raman scattering (SERS) from gold-coated silicon-germanium nanocone substrates that are decorated with 30-nm spherical gold nanoparticles (AuNPs). Finite-element simulations suggest that individual nanocones generate stronger electromagnetic enhancement with axial polarization (i.e., polarization parallel to the vertical axis of the nanocones) than with transverse polarization (i.e., polarization in the plane of the nanocone substrate), whereas the excitation in a typical Raman microscope is mainly polarized in the transverse plane. We introduce a practical approach to improve the SERS performance of the substrate by filling the valleys between nanocones with AuNPs. Simulations reveal an enhanced electric field at the nanoscale junctions formed between AuNPs and nanocones, and we explain this lateral coupling with a hybridization model for a particle-film system. We further experimentally verify the added enhancement by measuring SERS from trans-1,2-bi-(4-pyridyl) ethylene molecules absorbed onto the substrates. We report over one order-of-magnitude increase in SERS activities with the AuNP decoration (compared to the nanocone substrate without AuNPs) and achieve a spatially averaged enhancement factor of 1.78 x 10^8 at 785-nm excitation. Understanding and implementing the enhancing mechanism of structured metallic surfaces decorated with plasmonic nanoparticles open possibilities to substantially improve the SERS performance of the existing processengineered substrates.

We present a computational study of the plasmonic properties of gold-silica-gold multilayer nanoshells with the core offset from the center. Symmetry breaking, due to the core offset, makes plasmon resonances that are dark in concentric geometries visible. Applying plasmon hybridization theory, we explain the origin of these resonances from the interactions of an admixture of both primitive and multipolar modes between the core and the shell. The interactions introduce a dipole moment into the higher order modes and significantly enhance their coupling efficiency to light. To elucidate the symmetry breaking effect, we link the geometrical asymmetry to the asymmetrical distribution of surface charges and demonstrate illustratively the diminishing multipolar characteristic and increasing dipolar characteristic of the higher order modes. The relative amplitudes of the modes are qualitatively related by visual examination of the dipolar component in the surface charge distributions. Using polarization-dependent surface charge plots, we illustrate two distinct mode configurations despite their spectral similarities. We further demonstrate a trend of increasing absorption relative to scattering as the resonant wavelength red shifts in response to a larger core offset.

Aims: We propose and have experimentally studied a new method with improved sensitivity and specificity of imaging of living cells. Method: Intracellular photothermal bubbles generated around gold nanoparticles (NPs) and their clusters were proposed as optical scattering probes for the amplification of scattered light. Results: Microbubbles generated around gold spheres and shells with 10-ns 532-nm laser pulses in individual living cells (leukemia cells, lung and squamous carcinoma cancer cells) have amplified optical side scattering up to 1800-times relative to that of intracellular gold NPs, and without detectable damage to host cells. We explain the discovered optical amplification by the endocytosis-mediated clustering of NPs in cells, and by the selective generation of microbubbles (that do not disrupt the host cell) around these clusters at minimal levels of laser pulse fluence. Conclusions: Photothermal bubbles generated around laser-activated gold NPs may significantly improve the sensitivity and specificity of cell imaging, and can be considered as a new type of optical cellular probes.

The input impedance and radiation pattern of the inset-fed rectangular microstrip patch antenna are investigated. It is concluded that a shifted cosine-squared function describes well the variation of the resonant input resistance with the feed location (point of contact between the microstrip feed line and the notch) for a given patch and substrate geometry. The parameters of the shifted cosine-squared function depend on the notch width for a given patch and substrate geometry. The cross-polarization level is seen to increase as either the notch width or depth increases. However, for a fixed input resistance level, the notch depth decreases as the width increases, so that the cross-polarization level is not very sensitive to the notch width for a fixed input resistance.

Biomedical nanotechnology offers superior potential for diagnostic imaging of malignancy at the microscopic level. In addition to current research focused on dual-imaging and therapeutic applications in vivo, these novel particles may also prove useful for obtaining immediate diagnostic results in vitro at the patient bedside. However, translating the use of nanoparticles for cancer detection to point-of-care applications requires that conditions be optimized such that minimal time is needed for diagnostic results to become available. Thus far, no reports have been published on minimizing the time needed to achieve acceptable optical contrast of cancer cells incubated with nanoparticles. In this study, we demonstrate the use of gold nanoshells targeted to anti-HER2 antibodies that produce sufficient optical contrast with HER2-overexpressing SK-BR-3 breast cancer cells in only 5 min. This work validates the proof of concept that nanoshells targeted to extracellular biomarkers can be used to enhance cancer diagnostic imaging for use in point-of-care applications.

Optical trapping and micromanipulation has developed from an interesting novelty to a powerful and widely used tool, with the capability to move or trap microscopic live biological specimens and measure forces on the order of piconewtons, typical of forces in microbiological systems. Despite this, the range of particles typically trapped or manipulated is quite small, and it is unusual to see applications involving objects other than biological specimens or homogeneous isotropic microspheres, typically polymer or silica. However, particles can be modifled or specially fabricated to expand the possible applications of optical tweezers. For example, while non-absorbing homogeneous isotropic spheres cannot be rotated, optically anisotropic spheres can, and can therefore function as microscopic torque sen- sors, extending the usual translational micromanipulation and force measurement to rotational manipulation and torque sensing. The development of such particles has led to applications in microscale metrology and biophysics, along with potential deployment of optically-driven micro- machines in lab-on-a-chip devices. We present an overview of our work on the tailoring of microparticles for versatile optical trap- ping and micromanipulation. This includes approaches based on controlled chemistry | nano- assembly | and optical microfabrication. Beginning with the production of anisotropic vaterite microspheres, we review some of the applications, and di-culties encountered along the way. Some of these di-culties can be overcome by coating of the vaterite microspheres. We also discuss the use of anti-re∞ection coating to allow strong trapping of high refractive index parti- cles. The alternative strategy of producing arbitrarily shaped polymer microstructures through two-photon photopolymerization is also discussed. This can be used to produce optically-driven microrotors or structurally anisotropic microspheres to replace vaterites for particular applica- tions.

Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability.

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization.

Surface-enhanced Raman spectroscopy (SERS) is a known approach for detecting trace amounts of molecular species. Whereas SERS measurements have focused on enhancing the signal for sensing trace amounts of a chemical moiety, understanding how the substrate alters molecular Raman spectra can enable optical probing of analyte binding chemistry. Here we examine binding of trans-1,2-two(4- pyridyl) ethylene (BPE) to Au surfaces and understand variations in experimental data that arise from differences in how the molecule binds to the substrate. Monitoring differences in the SERS as a function of incubation time, a period of several hours in our case, reveals that the number of BPE molecules that chemically binds with the Au substrate increases with time. In addition, we introduce a direct method of accessing relative chemical enhancement from experiments that is in quantitative agreement with theory. The ability to probe optically specific details of metal/molecule interfaces opens up possibilities for using SERS in chemical analysis.

We have used short laser pulses to generate transient vapor nanobubbles around plasmonic nanoparticles. The photothermal, mechanical, and optical properties of such bubbles were found to be different from those of plasmonic nanoparticle and vapor bubbles, as well. This phenomenon was considered as a new complex nanosystem—plasmonic nanobubble (PNB). Mechanical and optical scattering properties of PNB depended upon the nanoparticle surface and heat capacity, clusterization state, and the optical pulse length. The generation of the PNB required much higher laser pulse fluence thresholds than the explosive boiling level and was characterized by the relatively high lower threshold of the minimal size (lifetime) of PNB. Optical scattering by PNB and its diameter (measured as the lifetime) has been varied with the fluence of laser pulse, and this has demonstrated the tunable nature of PNB.

Using a combination of first-principles theory and experiments, we provide a quantitative explanation for chemical contributions to surface-enhanced Raman spectroscopy for a well-studied organic molecule, benzene thiol, chemisorbed on planar Au(111) surfaces. With density functional theory calculations of the static Raman tensor, we demonstrate and quantify a strong mode-dependent modification of benzene thiol Raman spectra by Au substrates. Raman active modes with the largest enhancements result from stronger contributions from Au to their electron-vibron coupling, as quantified through a deformation potential, a well-defined property of each vibrational mode. A straightforward and general analysis is introduced that allows extraction of chemical enhancement from experiments for specific vibrational modes; measured values are in excellent agreement with our calculations., Comment: 5 pages, 4 figures and Supplementary material included as ancillary file

We develop a Lorenz-Mie solution for multilayer concentric spheres and present two separate studies on evaluating nanoshell spectra and anti-reflection coatings for improved optical trapping.

We demonstrate the potential of using nanoparticles for point of care cancer diagnostic imaging applications by achieving optical contrast between normal and cancerous epithelial cells at minimal incubation times through optimization of nanoshell-cell conjugations.

The goal of this paper is to study the dependence of resonant input resistance of the inset-fed patch on the notch and feed-line geometry. Different geometries, substrate materials, and feed lines were considered. A simple CAD formula for the input resistance is obtained. The effect of the notch on the radiation pattern is also studied to see how it influences cross polarization.

A practical way to adjust the input impedance of a rectangular patch antenna is to vary the feed position. The inset-fed patch provides a method of impedance control with a planar feed configuration. The goal of this paper is to further study the inset-fed patch in order to examine the dependence of the input impedance on the inset notch geometry, as well as to realize design tables that show the notch depth to obtain the best input match (minimum |S/sub 11/|) for the inset patch when using a 50 /spl Omega/ feed.

To the Editor: The recently invented technique of Bayesian localization microscopy1 relaxes the requirement for an extremely high signal-to-background ratio for single-molecule super-resolution microscopy2–5. It enables a standard wide-field fluorescence microscope to generate super-resolution images from the stochastic bleaching and blinking6,7 of fluorescence dyes (3B analysis). Although the new 3B microscopy has enormous potential to put the power of super-resolution imaging into the hands of average biologists, its underlying algorithm can be an impediment. Calculating an area of 50 × 50 pixels takes approximately a full day on a state-of-the-art desktop computer (Fig. 1a). For larger image areas, the need for massive computing resources makes the 3B analysis inaccessible to many labs and limits its scope of application. We report here a simple solution for everyone: the Amazon Elastic Compute Cloud (Amazon EC2). Figure 1 3B analysis using Amazon EC2 Aside from its flexible and cost-effective super-computing power, the greatest advantages of Amazon EC2 are its accessibility and ease of use. Normally, porting a computational code to a new system involves the configuration of a system-dependent software environment. The convoluted configuration process—not to mention the increased complexity for cluster computing—often discourages scientists without programming backgrounds. Use of Amazon EC2 significantly lowers this entry barrier. By packaging the executable program together with its running environment in a single image file known as the Amazon machine image (AMI), we can distribute 3B analysis to the public through the Amazon Marketplace. Hundreds of processes can run on instances, with each instance configured as an exact copy of the AMI. A user can launch massively parallel 3B processes on the Cloud from a local computer in just a few minutes (Fig. 1b and Supplementary Video 1). Large computing power allows data analysis without compromising on field of view. We tested Amazon EC2 using the photoactivatable (PA) mCherry–tagged podosome data from ref. 1 (Fig. 1c). The recorded image was divided into small mosaics of 12 × 12 pixels. The mosaics were analyzed in parallel and recombined to form a super-resolution image of 9.6 × 9.6 µm. Given the same computation time, an eight-thread 3.40-GHz Intel Core i7 desktop computer could analyze a region of 2.715 × 2.715 µm. The image generated from 100 virtual cores from Amazon EC2 was 12 times as large. An entry-level user of the Amazon Cloud may obtain up to 100 instances at about $0.02–$0.06 per instance hour. Depending on the market price, reconstruction of a 25 × 25–µm image costs less than $25. We strongly recommend the use of our bash scripts to continuously monitor the reconstructed image (Supplementary Software and tutorial therein). The calculation should be terminated only when the structrual features have converged, as otherwise potentially serious image artifacts may still be present. Researchers may apply for Amazon Web Service research grants for additional computing hours. We performed a test with 300 instances of 600 virtual cores. An image of PAmCherry-tagged tubulins in U2OS cells (from our own experiments) with the size of 150 pixels × 100 pixels × 1,500 frames could be reconstructed in only 210 min (Fig. 1d), whereas on a state-of-the-art desktop computer, this process would have taken 9 d. Cloud computing has been transforming the information technology industry through its elastic and flexible on-demand services. Although cloud computing has contributed to the success of many companies such as Dropbox and Netflix, until now it has been little used in biological research. Through this Correspondence, we hope to highlight its huge potential for the imaging community.

Metal nanoparticles (NPs) scatter and absorb light in precise, designable ways, making them agile candidates for a variety of biomedical applications. When NPs are introduced to a physiological environment and interact with cells, their physicochemical properties can change as proteins adsorb on their surface and they agglomerate within intracellular endosomal vesicles. Since the plasmonic properties of metal NPs are dependent on their geometry and local environment, these physicochemical changes may alter the NPs' plasmonic properties, on which applications such as plasmonic photothermal therapy and photonic gene circuits are based. Here we systematically study and quantify how metal NPs' optical spectra change upon introduction to a cellular environment in which NPs agglomerate within endosomal vesicles. Using darkfield hyperspectral imaging, we measure changes in the peak wavelength, broadening, and distribution of 100-nm spherical gold NPs' optical spectra following introduction to human breast adenocarcinoma Sk-Br-3 cells as a function of NP exposure dose and time. On a cellular level, spectra shift up to 78.6 ± 23.5 nm after 24 h of NP exposure. Importantly, spectra broaden with time, achieving a spectral width of 105.9 ± 11.7 nm at 95% of the spectrum's maximum intensity after 24 h. On an individual intracellular NP cluster (NPC) level, spectra also show significant shifting, broadening, and heterogeneity after 24 h. Cellular transmission electron microscopy (TEM) and electromagnetic simulations of NPCs support the trends in spectral changes we measured. These quantitative data can help guide the design of metal NPs introduced to cellular environments in plasmonic NP-mediated biomedical technologies.

The spectral and angular radiation properties of gold-silica-gold multilayer nanoshells are investigated using Mie theory for concentric multilayer spheres. The spectral tunability of multilayer nanoshells is explained and characterized by a plasmon hybridization model and a universal scaling principle. A thinner intermediate silica layer, scaled by particle size, red shifts the plasmon resonance. This shift is relatively insensitive to the overall particle size and follows the universal scaling principle with respect to the resonant wavelength of a conventional silica-gold core-shell nanoshell. The extra tunability provided by the inner core further shifts the extinction peak to longer wavelengths, which is difficult to achieve on conventional sub-100 nm nanoshells due to limitations in synthesizing ultrathin gold coatings. We found multilayer nanoshells to be more absorbing with a larger gold core, a thinner silica layer, and a thinner outer gold shell. Both scattering intensity and angular radiation pattern were found to differ from conventional nanoshells due to spectral modulation from the inner core. Multilayer nanoshells may provide more backscattering at wavelengths where silica-gold core-shell nanoshells predominantly forward scatter.

An antireflection coating improves the trapping of high-index particles in optical tweezers by reducing the scattering force. This can allow the strong trapping of high-index particles that cannot normally be trapped, and the use of lower numerical aperture objectives while still obtaining strong trapping. The improvement is not overly sensitive to the refractive index or thickness of the coating.