Contact angle measurements by confocal microscopy for non-destructive microscale surface characterization

Contact angle measurements are of great importance in surface characterization but the practical use has often been limited to macroscopic dimensions (millimeters). Therefore, we have developed a confocal microscopy method that allows non-destructive measurements of both low (<30 degrees ) and high (30 degrees -90 degrees ) contact angles. Low contact angles were measured by reconstructing the drop profile from the interference patterns in droplets condensed from atmospheric humidity. At higher contact angles water droplets with a small amount of fluorescein were sprayed onto the surfaces and 3D-image stacks were recorded and used to extract the contact angle. Suitable drop sizes were between a few up to about 50 mum radius, using a 40x magnification objective. Using drops >10 micrometers radius for microcontact angle measurements a good correlation was obtained between measured micro- and macrocontact angles. After microcontact angle measurements the surfaces were rinsed and heavy meromyosin motor fragments were adsorbed to the surface. Importantly, the sensitive actin propelling function of these motor proteins was not affected by the previous contact angle measurements using fluorescent droplets. This suggests that the methodology should be suitable for non-destructive characterization of different parts of micropatterned surfaces being developed for biological assays.     

Self-diffusion and collective diffusion of charged colloids studied by dynamic light scattering

A microemulsion of decane droplets stabilized by a nonionic surfactant film is progressively charged by substitution of a nonionic surfactant molecule by a cationic surfactant. We check that the microemulsion droplets remain identical within the explored range of volume fraction (0.02-0.18) and of the number of charges per droplet (0-40). We probe the dynamics of these microemulsions by dynamic light scattering. Despite the similar structures of the uncharged and charged microemulsions, the dynamics are very different. In the neutral microemulsion, the fluctuations of polarization relax, as is well-known, via the collective diffusion of the droplets. In the charged microemulsions, two modes of relaxation are observed. The fast one is ascribed classically to the collective diffusion of the charged droplets coupled to the diffusion of the counterions. The slow one has, to our knowledge, not been observed previously neither in similar microemulsions nor in charged spherical colloids. We show that the slow mode is also diffusive and suggest that its possible origin is the relaxation of local charge fluctuations via the local exchange of droplets bearing different numbers of charges. The diffusion coefficient associated with this mode is then the self-diffusion coefficient of the droplets.     

Measurement of mobility of adsorbed colloids by lateral force microscopy

The surface mobility of colloidal latex particles adsorbed on mica was measured by moving the particles with an AFM tip in the lateral force microscopy mode. The same particle was repeatedly scanned while the normal force was gradually increased, until the particle was displaced from its location on the substrate. The lateral (friction) force curve obtained for that scan was then used to determine the force needed to displace the particle. The data accumulated for approximately 100 particles indicate a wide distribution in the lateral force required. However, the data show that the mean lateral force is proportional to the particle diameter, with the effect of electrostatic interactions on the mobility of adsorbed particles seen to be weak. These results are consistent with classical theories of friction in macroscopic systems.     

Direct determination of interaction potentials from gas viscosity measurements alone

The direct inversion scheme of Smith and co-workers has been modified so as to remove the requirement that the well depth ? be known independently before the interaction potential can be determined from gas viscosity data. It is shown that the entire potential, including ?, can be determined within about 5% even when the viscosity measurements are uncertain by 1%.     

Study of the cholesterol-GM3 ganglioside interaction by surface pressure measurements and fluorescence microscopy

The nature of the cholesterol/glycolipid interaction in rafts being poorly understood, the interaction of cholesterol with the GM₃ ganglioside has been studied by surface pressure measurements and fluorescence microscopy. Results have been compared to those obtained with sphingomyelin (SM)-cholesterol and palmitoyl-oleoyl-phosphatidylcholine (POPC)–cholesterol monolayers. The analysis of (π–A) isotherms of mixed monolayers show a condensing effect of cholesterol on GM₃ molecules, in the same range than the effect observed with POPC and higher than the effect on SM. This is likely due to the similar state of GM₃ and POPC, since both molecules are in liquid expanded phases in our experimental conditions. The study of the cholesterol desorption induced by β-cyclodextrin suggests also that the GM₃–cholesterol interaction is rather weak as in the case of POPC–cholesterol interaction, and clearly lower than SM–cholesterol one. This lack of interaction is discussed in terms of nature of lipid chains and molecular shape, and suggests that no hydrogen bond is formed between GM₃ and cholesterol polar heads. Fluorescence microscopy performed on mixed GM₃–cholesterol monolayers shows the presence, at surface pressure higher than 10 mN/m, of particular blurring patterns without defined boundary, which could be due to a partial solubilization in one phase of different phases observed at lower surface pressure, whereas SM–cholesterol and POPC–cholesterol monolayers are homogeneous at the lateral resolution of our microscopy set-up.     

Direct visualization of colloidal rod assembly by confocal microscopy

The development of model materials and image processing methods to directly visualize and quantify colloidal rod assembly by means of confocal laser scanning microscopy (CLSM) is reported. Monodisperse fluorescent colloidal rods are prepared by the uniaxial extensional deformation of sterically stabilized microspheres at elevated temperatures. The particles are stably dispersed in refractive index matching mixed organic solvents for CLSM. An image processing algorithm is developed to detect rod backbones and extract particle centroids and orientation angles from the CLSM image volumes. By means of these methods we quantify the distribution of rod orientation angles in self-assembled structures of rods formed by sedimentation. We find the observations to be consistent with aspect-ratio-dependent jamming and orientational order/disorder transition in the rod sediments.     

Structure of polydispersed colloids characterised by light scattering and electron microscopy

The dynamics of growth and aggregation of colloidal silver iodide particles was followed by the static light scattering method. The particles were treated as spheres and they were stable in size in the defined time interval. This approach enabled the use of the Zimm plots in order to determine the radii of gyration and the radii of spherical particles. Stable AgI colloids, either positively or negatively charged, showed the usual Zimm diagrams, while the diagrams were untypical when the stability of the colloids decreased. The untypical Zimm diagrams showed 'curves' with envelopes and 'curves' with minima in the unstable domain and in the domain where the most rapid nucleation occurs, respectively. Satisfactory agreement of particle sizes within the limits of accuracy, determined using static--and dynamic light scattering data and of the values obtained from the electron microscopic images was shown. Fitting the theoretical and experimental data, P(theta) functions showed that the particle shapes approach the theoretical model for spheres and thin discs. The colloid stability of polydispersed aggregates was also explained using the second virial coefficient, its negative sign implying interaction of particles in the solution, its positive value indicating formation of new particles from the supernatant solution. In addition, the colloid stability can be characterised by the mass fractal dimension. For positive stable colloids, Dm = 2.70 +/- 0.26, it can be related to the reaction controlled processes, whereas for negative stable colloids, Dm = 1.97 +/- 0.19, it was attributed to the diffusion controlled processes.     

Investigations of the microscopic structure of poly(vinyl alcohol) hydrogels by confocal laser scanning microscopy

The fluorescence mode confocal laser scanning microscopy (CLSM) is introduced as an alternative method to investigate the bulk structure of poly(vinyl alcohol) (PVA) hydrogel. Investigations of the bulk structure of hydrogel samples, prepared by freezing and controlled thawing of aqueous PVA solutions followed by fluorochrome conjugation, were possible in the native state because with this technique water does not need to be removed prior to examination. This is of advantage to other methods, such as scanning electron microscopy, requiring dehydration by critical-point drying or freeze-etching, because both may result in a significant alteration of the gel structure. CLSM images of the hydrogel bulk structure were taken at several successive intervals from the surface into the hydrogel (up to 60 μm) without freeze-fracturing or cutting the sample. Detailed morphological characterization is achievable by superimposing series of images taken at successive intervals and by magnifying special regions of interest. Images of hydrogel bulk structures revealed a continuous, three-dimensional network that originates from phase-separation (spinodal decomposition) during the freezing period. The pore or mesh size in the cryogel increased, from about 2–7 μm, with decreasing PVA concentration. The surface layer was only a few microns thick, and the bulk structure underneath showed neither porosity gradients nor structural orientations. Received: 29 April 2000/Accepted: 18 August 2000     

Atomic force microscopy and magnetic force microscopy study of model colloids

Atomic force microscopy (AFM) is used to study the size, shape, and polydispersity of a variety of magnetic and nonmagnetic model colloids, previously imaged by transmission electron microscopy (TEM) only. Both height and phase images are analyzed and special attention is given to 3D morphology and softness of particles, as well as structures and presence of secondary components in the colloid, difficult to investigate with TEM. Several methods of tip characterization followed by deconvolution were applied in order to improve the accuracy of lateral diameter determination. In the case of magnetite particles dispersed in conventional ferrofluids, we explore both experimentally and theoretically the possibility of using magnetic force microscopy (MFM). We propose and discuss several models which allow to estimate the magnetic moment of a single domain superparamagnetic sphere using MFM, which cannot be done with other techniques; alternatively the tip magnetization can be determined.     

Study of the interaction of beta-cyclodextrin with phospholipid monolayers by surface pressure measurements and fluorescence microscopy

The interaction of beta-cyclodextrin (beta-CD) with different lipids has been studied, using Langmuir monolayers kept at constant surface pressure or constant spreading surface. Results show that beta-CD, injected beneath the monolayer, is able to desorb unsaturated palmitoyloleoylphosphatidylcholine (POPC) and sphingomyelin (SM) under specific experimental conditions. In this last case, SM monolayers, labeled with the fluorescent NBD-PC probe, were also observed by fluorescence microscopy, before and after beta-CD injection. Images show that SM monolayers are more homogeneous after beta-CD injection, because of the lipid desorption. At last, it seems that lipid desorption occurs only in a restricted surface pressure range, depending on the lipid.     

Calcium phosphate colloids with hierarchical structure controlled by polyaspartates

 Precipitation of calcium phosphate in a double-jet experiment in the presence of sodium polyaspartic acid results in a structural evolution of stable colloidal objects with a variety of unconventional morphologies. This structural evolution is characterized by a combination of techniques, namely static and dynamic light scattering, small-angle and wide-angle X-ray scattering, and transmission electron microscopy. It is shown that after immediate formation of amorphous precursor nanoparticles with a “parachute architecture”, the system transfers into a hollow sphere morphology in the 500-nm region and is composed of plateletlike nanocrystals, and again is transferred into a final, “snowball-like” morphology of high structural definition and monodispersity. The kinetics depends on the amount and the molecular weight of the polymer, but similar structural elements are found in all the cases examined. When repeated in a triple-jet experiment, which also ensures a constant polymer concentration, it was possible to intercept a postulated intermediate structure in practically complete yield, namely crystalline hydroxyapatite nanofibres with extremely high axial ratios which form an interwoven gel. Received: 11 May 2001 Accepted: 14 September 2001     

Study of dye diffusion in fibers by laser scanning confocal microscopy

The diffusion of a fluorescent dye in nylon 66 fibers was studied by using a laser scanning confocal microscope (LSCM) and by a conventional method. The diffusion coefficients were determined by both methods and they were in close agreement. The study of dye diffusion in fibers by LSCM is simple, quick and also able to provide high resolution images of fiber cross-sections and 3D images of the dye distribution in fibers. To our knowledge this is the first report of using a LSCM to study dye diffusion in polymeric fibers.     

Surfactant enhanced lipase containing films characterized by confocal laser scanning microscopy

An environmentally benign method for the synthesis of noble metal nanoparticles has been reported using aqueous solution of gum kondagogu (Cochlospermum gossypium). Both the synthesis, as well as stabilization of colloidal Ag, Au and Pt nanoparticles has been accomplished in an aqueous medium containing gum kondagogu. The colloidal suspensions so obtained were found to be highly stable for prolonged period, without undergoing any oxidation. SEM–EDXA, UV–vis spectroscopy, XRD, FTIR and TEM techniques were used to characterize the Ag, Au and Pt nanoparticles. FTIR analysis indicates that –OH groups present in the gum matrix were responsible for the reduction of metal cations into nanoparticles. UV–vis studies showed a distinct surface plasmon resonance at 412 and 525 nm due to the formation of Au and Ag nanoparticles, respectively, within the gum network. XRD studies indicated that the nanoparticles were crystalline in nature with face centered cubic geometry. The noble metal nanoparticles prepared in the present study appears to be homogeneous with the particle size ranging between 2 and 10 nm, as evidenced by TEM analysis. The Ag and Au nanoparticles formed were in the average size range of 5.5 ± 2.5 nm and 7.8 ± 2.3 nm; while Pt nanoparticles were in the size range of 2.4 ± 0.7 nm, which were considerably smaller than Ag and Au nanoparticles. The present approach exemplifies a totally green synthesis using the plant derived natural product (gum kondagogu) for the production of noble metal nanoparticles and the process can also be extended to the synthesis of other metal oxide nanoparticles.     

An approach to membrane fouling characterization by confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) is an optical microscopic technique that, among other advantages, can provide high-resolution images from different depths of a three-dimensional object, therefore rendering invasive techniques unnecessary for sample preparation. CSLM in fluorescence mode is a powerful technique in biological applications and in the microscopy of food materials. The main goal of the present study is to develop the appropriate strategies so that CSLM can be used for membrane fouling characterization during the filtration of protein solutions. Single and binary solutions of BSA–fluorescein and ovalbumin–Texas red conjugates were filtered using 0.8 μm polycarbonate membranes. Samples of the membranes at the end of the filtration runs were analyzed by CSLM. A standardized protocol for sample analysis by CSLM was developed and applied in this study. The most significant results show that CSLM can be used to visualize BSA–fluorescein and ovalbumin–Texas red conjugates on top of and inside the membranes, and that they can be distinguished when they jointly foul the membrane. Finally, if the appropriate sectioning is applied a 3D reconstruction of the membrane and the adsorbed/deposited protein can be obtained which give information on the fouling morphology.     

Calculation of vertical ionization potentials by configuration interaction

The ionization potentials of H₂O, N₂, C₂H₂, and HCN were calculated by frozen-orbital configuration interaction using doouble-zeta and double-zeta plus polarization functions basis sets. The results are compared to the observed values and to those from Rayleigh—Schrödinger perturbation theory. The effects of higher excitations are estimated and discussed.     

A new approach to non-destructive analysis of biofilms by confocal Raman microscopy

Confocal Raman microscopy (CRM) of biofilms enables one to determine the distribution of different microorganisms and other substances inside physiological intact microbial communities. These biofilms are of outstanding interest for biological wastewater treatment. In contrast to invasive techniques, such as fluorescent in situ hybridization (FISH), we were able to identify anaerobically ammonium-oxidising (anammox) bacteria without pretreatment processes of the samples just by its Raman vibrational signature. The presented results provide new insights into the complex interactions of different organisms in microbial communities without interfering with them.     

Pair interaction potentials with explicit polarization for molecular dynamics simulations of La(3+) in bulk water

Pair interaction potentials (IPs) were defined to describe the La(3+)-OH(2) interaction for simulating the La(3+) hydration in aqueous solution. La(3+)-OH(2) IPs are taken from the literature or parametrized essentially to reproduce ab initio calculations at the second-order Moller-Plesset level of theory on La(H(2)O)(8) (3+). The IPs are compared and used with molecular dynamics (MD) including explicit polarization, periodic boundary conditions of La(H(2)O)(216) (3+) boxes, and TIP3P water model modified to include explicit polarization. As expected, explicit polarization is crucial for obtaining both correct La-O distances (r(La-O)) and La(3+) coordination number (CN). Including polarization also modifies hydration structure up to the second hydration shell and decreases the number of water exchanges between the La(3+) first and second hydration shells. r(La-O) ((1))=2.52 A and CN((1))=9.02 are obtained here for our best potential. These values are in good agreement with experimental data. The tested La-O IPs appear to essentially account for the La-O short distance repulsion. As a consequence, we propose that most of the multibody effects are correctly described by the explicit polarization contributions even in the first La(3+) hydration shell. The MD simulation results are slightly improved by adding a-typically negative 1r(6)-slightly attractive contribution to the-typically exponential-repulsive term of the La-O IP. Mean residence times are obtained from MD simulations for a water molecule in the first (1082 ps) and second (7.6 ps) hydration shells of La(3+). The corresponding water exchange is a concerted mechanism: a water molecule leaving La(H(2)O)(9) (3+) in the opposite direction to the incoming water molecule. La(H(2)O)(9) (3+) has a slightly distorded "6+3" tricapped trigonal prism D(3h) structure, and the weakest bonding is in the medium triangle, where water exchanges take place.     

Time-resolved confocal fluorescence microscopy of trinitrobenzene-responsive organic nanofibers

Time-resolved confocal fluorescence microscopy is used to image and analyze quantitatively the influence of 1,3,5-trinitrobenzene on the fluorescence of organic nanofibers. These nanofibers are formed by self-assembly of 2,3-didecyloxyanthracene in methanol or from solutions drop-casted onto glass surfaces. Amplification of the fluorescence quenching emerges in the nanofibers as compared to the constituting monomer thus leading to efficient detection of nanomolar concentrations of TNB. The emission of dry nanofibers on glass is also efficiently quenched by vapors of TNB.     

Accuracy of energy extrapolation in multireference configuration interaction calculations

The accuracy of extrapolation procedures in conjunction with energy-based configuration selection in CI calculations is examined. The normally high accuracy of such extrapolation can deteriorate in multireference CI calculations when configuration functions of low weight are included in the root (reference) set. This is due to the inadequacy of second-order energy contribution estimates for the very large number of discarded low-contribution functions generated as single and double excitations from the minor members of the root set. The problem may be overcome by increasing the number of configurations included in the zero-order function used for the energy contribution estimation process. Illustrative results are presented for excited states of the H₂O molecule and the H₂O⁺ ion.