Atypical mobilities of single native DNA molecules in microchip electrophoresis revealed by differential interference contrast microscopy

A transmitted-light optical microscope using differential interference contrast (DIC) was employed to follow the real-time dynamics of different kb-sized single native dsDNA molecules without fluorescent-dye labeling. In a PDMS/glass microchip, the electrophoretic migration velocities of large dsDNA molecules are lower than small dsDNA molecules in a running buffer of 0.25% v/v nonionic polymeric surfactant C16E6 (n-alkyl polyoxyethylene ether) in 100 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer because the C16E6 behaved as a dynamic polymer. However, the order of migration reversed in 50 mM Gly-Gly buffer. The radial positions of individual DNA molecules (i.e., center or walls of the microchip) did not change the migration order. The atypical migration order correlated well with the results in CE. The alignment of the deformable molecules due to viscous drag is likely responsible for these observations.     

Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy

We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule–granule fusion before plasma membrane fusion was recorded. Electronic Supplementary Material Supplementary material is available for this article at     

Video enhanced differential interference contrast microscopy: Characterizing colloidal materials

Video enhanced differential interference contrast microscopy (VEDICM) permits an immediate, rapid characterization of association colloid aggregates and other colloidal aggregates by direct visualization on a television screen. Particles with sizes down to 500 A, their dynamics, fusion and slow flocculation can be directly pictured, recorded and analyzed in real time, freezeframe, slow motion or time lapse. It is precisely in the distance regime, 500–10,000 A, joining micellar chemistry to the field of biological structures, that classical techniques do have most difficulty. In this domain surfactant aggregates-vesicles, liposomes, microemulsions, microtubules-can exhibit an astonishing dynamic structural diversity and distribution of structures. These are highly sensitive to pH, salt, temperature, and surfactant concentration in ways which are partially understood at a theoretical level, but not formerly easily accessible.In this paper, the VEDICM technique is described and its ability to follow the spontaneous growth and fusion of vesicles upon changes in chemical environment is presented.Session lecture, Ninth International Conference on Non-Aqueous Solutions, Pittsburgh, PA, August 1984.     

Determining the full three-dimensional orientation of single anisotropic nanoparticles by differential interference contrast microscopy

Keeping track: By combining differential interference contrast (DIC) image pattern recognition with DIC polarization anisotropy, the exact full three-dimensional angular information of individual tilted gold nanorods positioned in the focal plane of the objective lens can be readily determined. The angular rotational modes and kinetics of individual in-focus gold nanorods can thus be resolved dynamically.     

Differential interference contrast microscopy for real-time dynamics and manipulation of single cells in microchannels

Nomarski differential interference contrast (DIC) microscopy was used for real-time dynamics of intact single cells in various microchannels for adaptation to microfluidic chip application. The cheek cell was chosen as a model, single cell and the dynamics was measured at the microchannels. The image resolution of single cell was shaper and more distinct in DIC than in conventional microscopy. The individual single living cells were also manipulated by both hydrodynamic and electrokinetic flow-driving forces at the microchannels. The DIC contrast was enhanced according to the order of round-, square-, and rectangle-type microchannels. The velocity of the single living cell was consistently increased with increasing electric field strength and pH. However, the velocity of cell was decreased with increasing run buffer concentration. The driving direction of the individual single cell was simply controlled by changing the polarity of the applied voltage and the electric field strength. The cells were consistently manipulated in the microchannel under the co-application of the low electric field of 2.44 V/cm, instead of the solo application of the hydrodynamic force.     

Determination of the number of cells in preimplantation embryos by using noninvasive optical quadrature microscopy in conjunction with differential interference contrast microscopy.

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.     

Endocytosis of a single mesoporous silica nanoparticle into a human lung cancer cell observed by differential interference contrast microscopy

The unique structural features of mesoporous silica nanoparticles (MSN) have made them very useful in biological applications, such as gene therapy and drug delivery. Flow cytometry, confocal microscopy, and electron microscopy have been used for observing the endocytosis of MSN. However, flow cytometry cannot directly observe the process of endocytosis. Confocal microscopy requires fluorescence labeling of the cells. Electron microscopy can only utilize fixed cells. In the present work, we demonstrate for the first time that differential interference contrast (DIC) microscopy can be used to observe the entire endocytosis process of MSN into living human lung cancer cells (A549) without fluorescence staining. There are three physical observables that characterize the locations of MSN and the stages of the endocytosis process: motion, shape, and vertical position. When it was outside the cell, the MSN underwent significant Brownian motion in the cell growth medium. When it was trapped on the cell membrane, the motion of the MSN was greatly limited. After the MSN had entered the cell, it resumed motion at a much slower speed because the cytoplasm is more viscous than the cell growth medium and the cellular cytoskeleton networks act as obstacles. Moreover, there were shape changes around the MSN due to the formation of a vesicle after the MSN had been trapped on the cell membrane and prior to entry into the cell. Finally, by coupling a motorized vertical stage to the DIC microscope, we recorded the location of the MSN in three dimensions. Such accurate 3D particle tracking ability in living cells is essential for studies of selectively targeted drug delivery based on endocytosis.     

Probe dependence of spatially heterogeneous dynamics in supercooled glycerol as revealed by single molecule microscopy

Spatially heterogeneous dynamics in supercooled glycerol over the temperature range 198 K (1.04T(g))-212 K (1.12T(g)) is investigated using widefield single molecule (SM) fluorescence microscopy. Measurements are performed using three different perylenedicarboximide probes to investigate whether probe size and probe-host interactions affect breadth of heterogeneity reported in the glassy host by such SM experiments. Rotational relaxation times of single probe molecules are measured, and for all probes, log-normal distributions of relaxation times are found. No significant change in relaxation time distribution as a function of temperature is evident for a given probe. However, across probes, probe rotational relaxation time is correlated with breadth of heterogeneous dynamics reported. Molecules that undergo changes in dynamics are identified using two complementary approaches that interrogate time scales between 10(3) and 10(6) τ(α), with τ(α) the structural relaxation time of glycerol. Exchange is found on the shortest time scales probed (~30 τ(c), with τ(c) the rotational correlation time of the probe) and is relatively temperature and probe independent. No evidence is found for additional exchange occurring on the longest time scales interrogated. Taken together with the fact that probes that rotate the fastest report the greatest breadth of spatially heterogeneous dynamics in the system, this indicates that exchange times reported from analysis of SM linear dichroism trajectories as described here are upper bounds on the average exchange time in the system.     

Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy

There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either l(o) or l(d) phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.     

Resolving rotational motions of nano-objects in engineered environments and live cells with gold nanorods and differential interference contrast microscopy

Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.     

Probing the structure of supported membranes and tethered oligonucleotides by fluorescence interference contrast microscopy

Fluorescence interference contrast microscopy (FLIC) is a powerful method to structurally characterize fluorescent objects with nanometer-scale resolution in the z direction. Here we use FLIC to characterize the water layer underlying supported membranes and membrane-tethered double-stranded oligonucleotides. FLIC measurements of supported membranes containing the lipid-anchored fluorescent dye DiI in both leaflets indicate the thickness of the water layer separating the solid support and the lower lipid leaflet is 1.3 +/- 0.2 nm. Addition of cobalt(II) chloride to a DiI-supported membrane quenches the fluorescence in the top leaflet of the supported membrane; FLIC measurements of this system precisely locate the DiI to the bottom leaflet. These experiments confirm the accuracy of the model and parameters used to determine the water layer thickness, demonstrate the ability to differentiate between fluorescent objects whose average position differs by approximately 1.9 nm, and provide a widely applicable method to test the resolution of other high-z-resolution fluorescent microscopies. FLIC measurements of Alexa-labeled double-stranded oligonucleotides tethered to a supported membrane indicate that the DNA double helix is oriented perpendicular to the surface. Complications that arise from uncertainly in the orientation of the fluorophore are discussed. Several improvements in FLIC methodology are described. These stringent tests of the resolution of FLIC and the ability to unambiguously determine fluorescent lipid distribution provide structural insight on assemblies at membrane interfaces and permit the detection of even subtle changes at such interfaces.     

Many-particle tracking with nanometer resolution in three dimensions by reflection interference contrast microscopy

We have developed and characterized a method, based on reflection interference contrast microscopy, to simultaneously determine the three-dimensional positions of multiple particles in a colloidal monolayer. To evaluate this method, the interaction of 6.8 microm (+/-5%) diameter lipid-derivatized silica microspheres with an underlying planar borosilicate substrate is studied. Measured colloidal height distributions are consistent with expectations for an electrostatically levitated colloidal monolayer. The precision of the method is analyzed using experimental techniques in addition to computational bootstrapping algorithms. In its present implementation, this technique achieves 16 nm lateral and 1 nm vertical precision.     

High-resolution differential interference contrast X-ray zone plates: Design and fabrication

Differential interference contrast is a potentially powerful technique for contrast enhancement in soft X-ray microscopy. We describe the design and fabrication of single-element diffractive optical elements suitable as objectives for high-resolution differential interference contrast microscopy in the water-window spectral range. A one-dimensional pattern calculation followed by an extension to two dimensions results in a pattern resolution of 1 nm, which is well below fabrication accuracy. The same fabrication process as for normal zone plates is applicable, but special care must be taken when converting the calculated pattern to a code for e-beam lithography.     

Colloidal adhesion of phospholipid vesicles: high-resolution reflection interference contrast microscopy and theory

High-resolution reflection interference contrast microscopy (HR-RICM) was developed for probing the deformation and adhesion of phospholipid vesicles induced by colloidal forces on solid surfaces. The new technique raised the upper limit of the measured membrane–substrate separation from 1 to 4.5 μm and improved the spatial resolution of the heterogeneous contact zones. It was applied to elucidate the effects of wall thickness, pH and osmotic stress on the non-specific adhesion of giant unilamellar vesicles (ULV) and multilamellar vesicles (MLV) on fused silica substrates. By simultaneous cross-polarization light microscopy and HR-RICM measurements, it was observed that ULV with the wall thickness of a single bilayer would be significantly deformed in its equilibrium state on the substrate as the dimension of its adhesive–cohesive zone was 29% higher than the theoretical value of a rigid sphere with the same diameter. Besides, electrostatic interaction was shown as a significant driving force for vesicle adhesions since the reduction in pH significantly increased the degree of deformation of adhering ULV and heterogeneity of the adhesion discs. The degree of MLV deformation on the solid surfaces was significantly less than that of ULV. When the wall thickness of vesicle increased, the dimension of contact zone was reduced dramatically due to the increase of membrane bending modulus. Most important, the adhesion strength of colloidal adhesion approached that of specific adhesion. Finally, the increase of osmotic stress led to the collapse of adhering vesicles on the non-deformable substrate and raised the area of adhesive contact zone. To interpret these results better, the equilibrium deformation of adhering vesicle was modeled as a truncated sphere and the adhesion energy was calculated with a new theory.     

Water molecule clusters measured at water/air interfaces using atomic force microscopy

During the tip approach to hydrophobic surfaces like the water/air interface, the measured interaction force reveals a strong attraction with a range of approximately 250 nm at some points along the interface. The range of this force is approximately 100 times larger than the measured for gold (approximately 3 nm) and 10 times larger than the one for hydrophobic silicon surfaces (approximately 25 nm). At other points the interface exerts a medium range repulsive force growing stepwise as the tip approaches the interface plane, consequently the hydrophobic force is a strong function of position. To explain these results we propose a model where the force on the tip is associated with the exchange of a small volume of the interface with a dielectric permittivity epsilon(int) by the tip with a dielectric permittivity epsilon(tip). By assuming a oscillatory spatial dependence for the dielectric permittivity it is possible to fit the measured force profiles. This dielectric spatial variation was associated with the orientation of the water molecules arrangement in the interfacial region. Small nanosized hydrogen-bond connected cages of water molecules present in bulk water at the interface are oriented by the interfacial electric field generated by the water molecules broken bonds, one broken hydrogen bond out of every four. This interfacial field orients small clusters formed by approximately 100 water molecules into larger clusters (approximately 100 nm). In the limit of small (less than 5 nm thick) water molecule cages we have modeled the static dielectric permittivity (epsilon) as the average response of those cages. In these regions the dielectric permittivity for water/air interfaces decreases monotonically from the bulk value epsilon approximately 80 to approximately 2 at the interface. For regions filled with medium size cages, the tip senses the structure of each cage and the static dielectric permittivity is matched to the geometrical features of these cages sized approximately 25 to 40 nm. Interfacial electric energy density values were calculated using the electric field intensity and the dielectric permittivity obtained by the fitting of the experimental points. The integration of the electric energy density along the interfacial region gives a value of 0.072 J m(-2) for interfacial energy density for points where the hydrophobic force has a range of approximately 250 nm. Regions formed by various clusters result in lower values of the interfacial energy density.     

The dynamic nature of contact angles as measured by atomic force microscopy

Atomic force microscopy appears to be a useful tool for determining the contact angle for small particles. It is shown in this paper that the contact angle of a spherical polyethylene particle changes with the speed of the AFM piezoelectric translator. Such dynamic behavior of the contact angle and other uncertainties such as the position of the three-phase contact on the particle surface during bubble-particle interaction make it difficult to decide whether or not the AFM single-particle contact angle can be used to describe the hydrophobic state of the particle surface.     

Ferrocenylundecanethiol self-assembled monolayer charging correlates with negative differential resistance measured by conducting probe atomic force microscopy

Electrical and mechanical properties of metal-molecule-metal junctions formed between Au-supported self-assembled monolayers (SAMs) of electroactive 11-ferrocenylundecanethiol (FcC(11)SH) and a Pt-coated atomic force microscope (AFM) tip have been measured using a conducting probe (CP) AFM in insulating alkane solution. Simultaneous and independent measurements of currents and bias-dependent adhesion forces under different applied tip biases between the conductive AFM probe and the FcC(11)SH SAMs revealed reversible peak-shaped current-voltage (I-V) characteristics and correlated maxima in the potential-dependent adhesion force. Trapped positive charges in the molecular junction correlate with high conduction in a feature showing negative differential resistance. Similar measurements on an electropassive 1-octanethiol SAM did not show any peaks in either adhesion force or I-V curves. A mechanism involving two-step resonant hole transfer through the occupied molecular orbitals (MOs) of ferrocene end groups via sequential oxidation and subsequent reduction, where a hole is trapped by the phonon relaxation, is proposed to explain the observed current-force correlation. These results suggest a new approach to probe charge-transfer involving electroactive groups on the nanoscale by measuring the adhesion forces as a function of applied bias in an electrolyte-free environment.     

Differential interference contrast polarization anisotropy for tracking rotational dynamics of gold nanorods

We describe differential interference contrast (DIC) polarization anisotropy for tracking rotational dynamics of gold nanorod (AuNR) probes. DIC polarization anisotropy enabled us to reveal the unidirectional clockwise circular translocation of an AuNR attached to a kinesin-driven microtubule and to precisely determine the real-time orientation of the AuNR during the dynamic process.     

Single molecule charging by atomic force microscopy

AFM/KPM charging and charge mapping of polyamine charge carriers in a PMMA matrix is reported. Selective charging of the designed charge carrier is demonstrated at concentrations down to a single molecule. This works constitutes electrochemical charging and detection of single redox-active organic molecules in low dielectric matrices by probe microscopy.     

Synthesis of novel fluorescent molecule and its polymeric form with aniline as fluorescent and supercapacitor electrode materials

In this study, a fluorescent material, 2‐naphthyl‐4‐amino benzoate, is synthesized by the esterification of 4‐aminobenzoic acid with 2‐naphthol. This molecule is used in the bulk polymerization of aniline, which results in the formation of poly(aniline‐2‐naphthyl‐4‐aminobenzoate). For comparison, polyaniline and also poly(aniline‐4‐aminobenzoic acid) salts are prepared via bulk polymerization. Formation and properties of these polymeric materials are evaluated by Fourier‐transform infrared (FT‐IR), ¹³C nuclear magnetic resonance, matrix‐assisted laser desorption ionization, UV‐Vis, Fluorescence, X‐ray diffraction (XRD), Field emission‐scanning electron microscopy (FE‐SEM), Differential scanning calorimetry (DSC), thermogravimetric analysis, electrical resistance and electrochemical techniques. P(ANI‐2NA4ABA) is obtained in nanofiber morphology in 106 wt% yield with respect to the amount of aniline used with comparable conductivity of conventional polyaniline salts. This polymer salt is stable up to 220°C and indicates melting at 146°C on heating and crystal formation at 128°C on cooling. This polymer shows higher wavelength fluorescence compared to the conventional polyaniline salts. This polymer is used as an electrode material without binder, which shows a specific capacitance of 360 F g^?1 at 0.25 A g^?1.