Dark-field Optical Microscopy Research Articles

Polymeric plasticizer extends the lifetime of PVC-membrane ion-selective electrodes

The nature of the plasticizer plays a pivotal role in the analytical performance of polymer membrane ion sensors. Conventional plasticizers suffer leaching or migration from the membrane and exudation, both of which could limit the lifetime of sensors based on plasticized membranes. Herein, we describe the use of polyester sebacate (PES), a model polymeric plasticizer, in the preparation of poly (vinyl chloride) (PVC) membrane ion-selective electrodes (ISEs) using valinomycin as ionophore. PVC membrane electrodes plasticized with polyester sebacate demonstrated potentiometric response characteristics that compared favorably to ones plasticized with the conventional and similarly structured plasticizer bis(2-ethylhexyl) sebacate (DOS). Increasing the content of polyester sebacate in the membrane enhanced the response and improved the selectivity of valinomycin-based ISEs toward potassium over sodium. Various methods, including electrochemical impedance spectroscopy, UV-vis spectroscopy, dark field optical microscopy, and potentiometry were employed to study the effect of plasticizer on the leaching of the membrane components and the lifetime of both DOS- and PES-plasticized membranes. PES-plasticized electrodes maintained Nernstian response and high selectivity for more than four months, an improvement over DOS-plasticized membrane electrodes. This was attributed to enhanced retention of the membrane components because of the high polymeric nature of the polyester sebacate. These characteristics suggest that polyester sebacate is a good candidate to replace the conventional plasticizers in preparing PVC membrane electrodes with longer lifetime.

Localized surface plasmon resonance (LSPR) biosensing using gold nanotriangles: detection of DNA hybridization events at room temperature.

We present a proof-of-concept of the application of gold nanotriangles in sequence specific DNA detection, using localized surface plasmon resonance (LSPR) spectroscopy and dark-field optical microscopy. The sensing platform comprises gold nanotriangles immobilized on a glass chip and oligonucleotides as probes. Probe formation and testing complementary and non-complementary targets followed common chip technology protocols. Gold nanotriangles showed a remarkable sensitivity of 468 nm per RIU and allowed detection of 20-mer targets. When the target sequence was part of a 50-mer synthetic DNA oligonucleotide, LSPR shifts as high as 35 nm were observed. Conversely, when the target was present in PCR products of ca. 350 bp, obtained from clinical samples, LSPR shifts larger than 20 nm were observed. Moreover, LSPR shifts were less than ±1 nm for the respective non-complementary targets. These results with gold nanotriangles as sensors are a notable improvement to the LSPR shifts of less than 5 nm usually obtained for spherical gold nanoparticles of comparable sizes. Optimal conditions for the detection of synthetic and PCR product targets using gold nanotriangles and oligonucleotide probes were achieved with low percentages of intercalating thioalkanes; target hybridization at room temperature, 3 hours of incubation, and 2× SSC buffer stringency conditions.

Hyperspectral fluorescence imaging for cellular iron mapping in thein vitromodel of Parkinson’s disease

Parkinson's disease (PD) is characterized by progressive dopaminergic cell loss in the substantia nigra (SN) and elevated iron levels demonstrated by autopsy. Direct visualization of iron with live imaging techniques has not yet been successful. The aim of this study is to visualize and quantify the distribution of cellular iron using an intrinsic iron hyperspectral fluorescence signal. The 1-methyl-4-phenylpyridinium (MPP+)-induced cellular model of PD was established in SHSY5Y cells exposed to iron with ferric ammonium citrate (FAC, 100 μM). The hyperspectral fluorescence signal of iron was examined using a high-resolution dark-field optical microscope system with signal absorption for the visible/near infrared spectral range. The 6-h group showed heavy cellular iron deposition compared with the 1-h group. The cellular iron was dispersed in a small particulate form, whereas the extracellular iron was aggregated. In addition, iron particles were found to be concentrated on the cell membrane/edge of shrunken cells. The iron accumulation readily occurred in MPP+-induced cells, which is consistent with previous studies demonstrating elevated iron levels in the SN. This direct iron imaging could be applied to analyze the physiological role of iron, and its application might be expanded to various neurological disorders involving metals, such as copper, manganese, or zinc.

Non-destructive and rapid evaluation of chemical vapor deposition graphene by dark field optical microscopy

Non-destructive and rapid evaluation of graphene directly on the growth substrate (Cu foils) by dark field (DF) optical microscopy is demonstrated. Without any additional treatment, graphene on Cu foils with various coverages can be quickly identified by DF imaging immediately after chemical vapor deposition growth with contrast comparable to scanning electron microscopy. The improved contrast of DF imaging compared to bright field optical imaging was found to be due to Rayleigh scattering of light by the copper steps beneath graphene. Indeed, graphene adlayers are readily distinguished, due to the different height of copper steps beneath graphene regions of different thickness.

Study of Charge-Dependent Transport and Toxicity of Peptide-Functionalized Silver Nanoparticles Using Zebrafish Embryos and Single Nanoparticle Plasmonic Spectroscopy

Nanomaterials possess unusually high surface area-to-volume ratios and surface-determined physicochemical properties. It is essential to understand their surface-dependent toxicity in order to rationally design biocompatible nanomaterials for a wide variety of applications. In this study, we have functionalized the surfaces of silver nanoparticles (Ag NPs, 11.7 ± 2.7 nm in diameter) with three biocompatible peptides (CALNNK, CALNNS, CALNNE) to prepare positively (Ag-CALNNK NPs(+ζ)), negatively (Ag-CALNNS NPs(-2ζ)), and more negatively charged NPs (Ag-CALNNE NPs(-4ζ)), respectively. Each peptide differs in a single amino acid at its C-terminus, which minimizes the effects of peptide sequences and serves as a model molecule to create positive, neutral, and negative charges on the surface of the NPs at pH 4-10. We have studied their charge-dependent transport into early developing (cleavage-stage) zebrafish embryos and their effects on embryonic development using dark-field optical microscopy and spectroscopy (DFOMS). We found that all three Ag-peptide NPs passively diffused into the embryos via their chorionic pore canals, and stayed inside the embryos throughout their entire development (120 h), showing charge-independent diffusion modes and charge-dependent diffusion coefficients. Notably, the NPs create charge-dependent toxic effects on embryonic development, showing that the Ag-CALNNK NPs(+ζ) (positively charged) are the most biocompatible while the Ag-CALNNE NPs(-4ζ) (more negatively charged) are the most toxic. By comparing with our previous studies of the same sized citrated Ag and Au NPs, the Ag-peptide NPs are much more biocompatible than the citrated Ag NPs, and nearly as biocompatible as the Au NPs, showing the dependence of nanotoxicity upon the surface charges, surface functional groups, and chemical compositions of the NPs. This study also demonstrates powerful applications of single NP plasmonic spectroscopy for quantitative analysis of single NPs in vivo and in tissues, and reveals the possibility of rational design of biocompatible NPs.

Multiple Membrane Interactions and Versatile Vesicle Deformations Elicited by Melittin

Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the “increasing membrane area”, “phased shrinkage”, or “solubilization” of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, β-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions.

Influence of Gas Phase Equilibria on the Chemical Vapor Deposition of Graphene

We have investigated the influence of gas phase chemistry on the chemical vapor deposition of graphene in a hot wall reactor. A new extended parameter space for graphene growth was defined through literature review and experimentation at low pressures (≥0.001 mbar). The deposited films were characterized by scanning electron microscopy, Raman spectroscopy, and dark field optical microscopy, with the latter showing promise as a rapid and nondestructive characterization technique for graphene films. The equilibrium gas compositions have been calculated across this parameter space. Correlations between the graphene films grown and prevalent species in the equilibrium gas phase revealed that deposition conditions associated with a high acetylene equilibrium concentration lead to good quality graphene deposition, and conditions that stabilize large hydrocarbon molecules in the gas phase result in films with multiple defects. The transition between lobed and hexagonal graphene islands was found to be linked to the concentration of the monatomic hydrogen radical, with low concentrations associated with hexagonal islands.

The effect of magnetically induced linear aggregates on proton transverse relaxation rates of aqueous suspensions of polymer coated magnetic nanoparticles

It has been recently reported that for some suspensions of magnetic nanoparticles the transverse proton relaxation rate, R(2), is dependent on the time that the sample is exposed to an applied magnetic field. This time dependence has been linked to the formation of linear aggregates or chains in an applied magnetic field via numerical modeling. It is widely known that chain formation occurs in more concentrated ferrofluids systems and that this has an affect on the ferrofluid properties. In this work we examine the relationships between colloidal stability, the formation of these linear structures, and changes observed in the proton transverse relaxation rate of aqueous suspensions of magnetic particles. A series of iron oxide nanoparticles with varying stabilizing ligand brush lengths were synthesized. These systems were characterized with dynamic light scattering, transmission electron microscopy, dark-field optical microscopy, and proton transverse relaxation rate measurements. The dark field optical microscopy and R(2) measurements were made in similar magnetic fields over the same time scale so as to correlate the reduction of the transverse relaxivity with the formation of linear aggregates. Our results indicate that varying the ligand length has a direct effect on the colloidal arrangement of the system in a magnetic field, producing differences in the rate and size of chain formation, and hence systematic changes in transverse relaxation rates over time. With increasing ligand brush length, attractive inter-particle interactions are reduced, which results in slower aggregate formation and shorter linear aggregate length. These results have implications for the stabilization, characterization and potentially the toxicity of magnetic nanoparticle systems used in biomedical applications.

Probing the Role of Rotational Dynamics in Cellular Transport

While it can generally be said that cellular function is critically dependent on the fidelity of cargo transport, processive transport is even more important in the axons and dendrites of neurons, where a cell must regulate populations of molecules on length scales that can range up to meters. Consequently, much effort has been made to investigate the translocation of cargoes in neurons and the properties of the motors responsible therein. Though biocompatible fluorophores have become increasingly powerful tools for study of motor-driven transport, they suffer from photobleaching and require bright illumination which can be toxic to live cells. Most conventional fluorescent approaches are further limited by the lack of oreintation information they provide. On the other hand, with the small diameter of neurites and the high levels of traffic they support through a crowded environment, orientation of the cargoes relative to the cytoskeletal tracks they are moving on can be vital. We present an experimental approach making use of dark field optical microscopy and gold nanorods as reporters of both lateral translocation as well as orientation of cargo in neurons. using relatively low illumination intensity, we can measure dynamics of single cargoes moving in the image plane and resolve changes in the azimuth and polar angles all at millisecond time scales. Furthermore, the gold nanorods can be specifically delivered to the cell body or axon terminal by culturing the neurons in microfluidic devices with separate chambers, enabling the investigator to resolve differences between retrograde and anterograde transport. The ability to track axonal transport with a high temporal and spatial dynamic range reveals several kinds of orientational changes of moving cargoes that correlate with transport dynamics, allowing more detailed inferences into changes in the activity of molecular motors.

Energetic-Assisted Scanning Thermal Lithography for Patterning Silver Nanoparticles in Polymer Films

Energetic-assisted scanning thermal lithography (SThL) was demonstrated with the addition of benzoyl peroxide (BPO) for patterning silver nanoparticles. SThL samples were prepared by spin-coating poly(methyl methacrylate) (PMMA) thin films preloaded with BPO and silver nitrate precursors. Localized thermal analysis via probe heating demonstrated that the BPO decomposition in the polymer film took place at the temperature of 80 °C. Above this temperature, the thermal probe initiated the decomposition of the peroxide, which resulted in the in situ discharge of exothermal energy to compensate the joule shortage and the rapid cooling in the SThL thin film samples. The additional joule energy thermally enhanced the synthesis of silver nanoparticles, which were patterned and embedded in the PMMA thin film. Surface plasmon resonance scattering of these silver nanoparticles was observed by dark-field optical microscopy, whereas the nanoparticle distribution was examined by transmission electron microscopy. Variations in the scanning probe temperatures and peroxide concentrations were carefully investigated to optimize the thermal lithography efficiency upon the addition of energetics.

Physicochemical Analysis from Real-Time Imaging of Liposome Tubulation Reveals the Characteristics of Individual F-BAR Domain Proteins

The Fer-CIP4 homology-BAR (F-BAR) domain, which was identified as a biological membrane-deforming module, has been reported to transform lipid bilayer membranes into tubules. However, details of the tubulation process, the mechanism, and the properties of the generated tubules remain unknown. Here, we successfully monitored the entire process of tubulation and the behavior of elongated tubules caused by four different F-BAR domain family proteins (FBP17, CIP4, PSTPIP1, and Pacsin2) using direct real-time imaging of giant unilamellar liposomes with dark-field optical microscopy. FBP17 and CIP4 develop many protrusions simultaneously over the entire surface of individual liposomes, whereas PSTPIP1 and Pacsin2 develop only a few protrusions from a narrow restricted part of the surface of individual liposomes. Tubules formed by FBP17 or CIP4 have higher bending rigidities than those formed by PSTPIP1 or Pacsin2. The results provide striking evidence that these four F-BAR domain family proteins should be classified into two groups: one group of FBP17 and CIP4 and another group of PSTPIP1 and Pacsin2. This classification is consistent with the phylogenetic proximity among these proteins and suggests that the nature of the respective tubulation is associated with biological function. These findings aid in the quantitative assessment with respect to manipulating the morphology of lipid bilayers using membrane-deforming proteins.

Fundamental mechanism for electrically actuated mechanical resonances in ZnO nanowhiskers

In this study, we elucidate the fundamental mechanism for electrically actuated mechanical resonances in semiconducting ZnO nanowhiskers (NWs). Based on visual detection and measurement of mechanical resonances in ZnO NWs using a scanning electron microscope (SEM), previous studies have attributed dynamic charge induction as the fundamental mechanism for the observed resonances. We show that the use of an electron beam as a resonance detection tool alters the intrinsic electrical character of the ZnO, and makes it difficult to identify the source of the charge necessary for the electrostatic actuation. A systematic study of the amplitude of electrically actuated as-grown and gold-coated ZnO NWs in the presence (absence) of an electron beam using a SEM (dark-field optical microscope) suggest that our ZnO NWs intrinsically support static charge actuation.

Tunable enhancement of light absorption and scattering in Si1−xGexnanowires

Semiconducting nanowires (NWs) are good candidates for new optoelectronic or photovoltaic devices due to their excellent ability to guide, scatter, or absorb light, from near ultraviolet to near infrared. The existence of morphology-dependent optical resonances opens a promising route to overcome the intrinsic limitations of the constituent material and optimize their interaction with light. We propose a thorough investigation of the optical properties of Si${}_{1\ensuremath{-}x}$Ge${}_{x}$ alloy nanowires addressing the influence of NW diameter, composition, light polarization, and angle of incidence on their scattering and absorption. Our results clearly show that the Ge composition provides an additional degree of freedom to tailor the optical response of these one-dimensional nano-objects and that resonant enhancement of both absorption and scattering can be obtained in the infrared range at relatively small diameters compared to pure Si nanowires. These results are confirmed by complementary light scattering and Raman spectroscopy experiments using confocal dark field optical microscopy on individual nanowires fabricated by Au-catalyzed vapor-liquid-solid synthesis.

A New Approach to Assess Gold Nanoparticle Uptake by Mammalian Cells: Combining Optical Dark‐Field and Transmission Electron Microscopy

Toxicological effects of nanoparticles are associated with their internalization into cells. Hence, there is a strong need for techniques revealing the interaction between particles and cells as well as quantifying the uptake at the same time. For that reason, herein optical dark-field microscopy is used in conjunction with transmission electron microscopy to investigate the uptake of gold nanoparticles into epithelial cells with respect to shape, stabilizing agent, and surface charge. The number of internalized particles is strongly dependent on the stabilizing agent, but not on the particle shape. A test of metabolic activity shows no direct correlation with the number of internalized particles. Therefore, particle properties besides coating and shape are suspected to contribute to the observed toxicity.

Plasmon Spectroscopy and Imaging of Individual Gold Nanodecahedra: A Combined Optical Microscopy, Cathodoluminescence, and Electron Energy-Loss Spectroscopy Study

Imaging localized plasmon modes in noble-metal nanoparticles is of fundamental importance for applications such as ultrasensitive molecular detection. Here, we demonstrate the combined use of optical dark-field microscopy (DFM), cathodoluminescence (CL), and electron energy-loss spectroscopy (EELS) to study localized surface plasmons on individual gold nanodecahedra. By exciting surface plasmons with either external light or an electron beam, we experimentally resolve a prominent dipole-active plasmon band in the far-field radiation acquired via DFM and CL, whereas EELS reveals an additional plasmon mode associated with a weak dipole moment. We present measured spectra and intensity maps of plasmon modes in individual nanodecahedra in excellent agreement with boundary-element method simulations, including the effect of the substrate. A simple tight-binding model is formulated to successfully explain the rich plasmon structure in these particles encompasing bright and dark modes, which we predict to be fully observable in less lossy silver decahedra. Our work provides useful insight into the complex nature of plasmon resonances in nanoparticles with pentagonal symmetry.

Magnetic light.

Spherical silicon nanoparticles with sizes of a few hundreds of nanometers represent a unique optical system. According to theoretical predictions based on Mie theory they can exhibit strong magnetic resonances in the visible spectral range. The basic mechanism of excitation of such modes inside the nanoparticles is very similar to that of split-ring resonators, but with one important difference that silicon nanoparticles have much smaller losses and are able to shift the magnetic resonance wavelength down to visible frequencies. We experimentally demonstrate for the first time that these nanoparticles have strong magnetic dipole resonance, which can be continuously tuned throughout the whole visible spectrum varying particle size and visually observed by means of dark-field optical microscopy. These optical systems open up new perspectives for fabrication of low-loss optical metamaterials and nanophotonic devices.

High-throughput ultrasensitive characterization of chemical, structural and plasmonic properties of EBL-fabricated single silver nanoparticles

Electron beam lithography (EBL) has become a popular means to prepare a wide variety of nano-arrays for numerous studies and applications, including photonics and sensors. Their fabrications and characterizations are costly and time consuming, underscoring the importance of developing effective tools to rapidly study their physicochemical stabilities and properties over time. In this study, we characterized EBL-fabricated single silver nanoparticle (Ag NP) arrays over their 12-week exposure to ambient conditions using SEM/EDS, AFM and dark-field optical microscopy and spectroscopy (DFOMS). We found that chemical compositions, structural morphologies and plasmonic optical properties of single NPs altered drastically over the exposure. Single cuboid and triangular-prism Ag NPs degraded at rates of (0.74 ± 0.02) and (0.66 ± 0.02) per week, and their localized surface plasmon resonance (LSPR) spectra showed striking blue-shifts (171 ± 25 and 203 ± 35 nm) over the 12-week exposure, respectively. Plasmonic colors of single NPs changed distinctively from red to green over the 12-week exposure. The LSPR spectra of individual NPs in each array were acquired simultaneously and correlated specifically with their SEM and AFM images, demonstrating that DFOMS can serve as high-throughput, ultrasensitive and non-invasive means to characterize chemical, structural and optical properties of nano-arrays in situ in real time at single-NP resolution.

Single nanoparticle spectroscopy for real-time in vivo quantitative analysis of transport and toxicity of single nanoparticles in single embryos

Nanomaterials exhibit distinctive physicochemical properties and promise a wide range of applications from nanotechnology to nanomedicine, which raise serious concerns about their potential environmental impacts on ecosystems. Unlike any conventional chemicals, nanomaterials are highly heterogeneous, and their properties can alter over time. These unique characteristics underscore the importance of study of their properties and effects on living organisms in real time at single nanoparticle (NP) resolution. Here we report the development of single-NP plasmonic microscopy and spectroscopy (dark-field optical microscopy and spectroscopy, DFOMS) and ultrasensitive in vivo assay (cleavage-stage zebrafish embryos, critical aquatic species) to study transport and toxicity of single silver nanoparticles (Ag NPs, 95.4 ± 16.0 nm) on embryonic developments. We synthesized and characterized purified and stable (non-aggregation) Ag NPs, determined their sizes and doses (number), and their transport mechanisms and effects on embryonic development in vivo in real time at single-NP resolution. We found that single Ag NPs passively entered the embryos through their chorionic pores via random Brownian diffusion and stayed inside the embryos throughout their entire development (120 h), suggesting that the embryos can bio-concentrate trace NPs from their environment. Our studies show that higher doses and larger sizes of Ag NPs cause higher toxic effects on embryonic development, demonstrating that the embryos can serve as ultrasensitive in vivo assays to screen biocompatibility and toxicity of the NPs and monitor their potential release into aquatic ecosystems.

Real time plasmonic spectroscopy of the interaction of Hg2+ with single noble metal nanoparticles

In this work, we use dark-field optical microscopy (DFM) and hyper spectral imaging (HSI) to study the interactions of single Ag and Au nanoparticles (NPs) with Hg2+ in real time, at room temperature (25 °C). NPs were immobilized on glass substrates using 3-aminopropyltrimethoxysilane (APTMS) as the anchoring agent. Red, green and blue colors were assigned to the particles in hyper spectral images on the basis of their relative scattering intensities at 640, 550 and 460 nm, where the particles showed maxima in their scattering spectra. While Ag NPs showed all the colors, Au NPs were mostly red and rarely green in DFM images. The scattering spectra of Ag NPs were more blue shifted (with an average shift of 46 nm in the case of red particles) in a given time compared to Au NPs, after passing over Hg2+ solution and these shifts increased with time. Depending on the extent of blue shift, the colors of the particles got modified. Red particles appeared more reactive than green and blue, as revealed from the larger extent of shifts and their time dependence. The greater reactivity of red particles is attributed to their anisotropic nature possessing reactive tips, edges and more surface area due to their large size. The effect of quality of water on the scattering spectrum was checked by passing over deionized (DI) and tap waters separately, which showed that the effect is minimal compared to the presence of Hg2+, when data at a given time, flow rate and temperature were compared. Solution phase interactions of NPs with Hg2+ were also performed for comparison. These were characterized by UV-vis absorption spectroscopy, transmission electron microscopy (TEM) and energy dispersive analysis of X-rays (EDAX). Solution phase experiments showed citrate-induced aggregation of Ag NPs and partial reduction of Hg2+ to Hg0 upon exposure to Hg2+. Immobilized particles cannot aggregate and they show only reduction.

Three-Dimensional Real-Time Imaging of Gliding Motility in Apicomplexan Parasites using 4D Dark Field Microscopy

The speed and sensitivity of detection methods along with the efficiency of the process or signal representing the sample delimit optical imaging of living cells. To facilitate the imaging of living biological cells in real time in three dimensions, and in their native contrast, we have developed an imaging method composed of a dark field optical microscope coupled with a high-speed CMOS camera capable of capturing images at 5,000 fps at full field, equipped with a motorized assembly regulating the position of the objective. We have successfully applied the instrument to the imaging of 200nm gold particles and also to studying motility in the Apicomplexan parasite Neospora caninum. N. caninum is a close relative of the pathogenic apicomplexans Toxoplasma gondii (which causes disease in immunocompromised individuals) and Plasmodium falciparum (the causative agent of malaria), which exhibit the same characteristic dynamic motion. Apicomplexan parasites rely on apical constriction to lead the parasite in twirling, helical, and clockwise gliding motion for motility on solid surfaces and also to invade host cell membranes. The precise penetrative technique of these organisms has been little understood due to current restrictions in imaging, though it has been posited that more in-depth knowledge of the gliding motility can uncover the invasion mechanism. We are currently capable of successfully capturing 3D volumes of active N. caninum cells at 50-100Hz with a 5-20μm vertical transverse range. We employ a Fourier-based deconvolution algorithm to reduce noise and better visualize the 4D data, which is expected to facilitate the identification of features in N. caninum cells that are critical for its mechanism of invasion.