A study of localised galvanic replacement of copper and silver films with gold using scanning electrochemical microscopy

Galvanic replacement represents a highly significant process for the fabrication of bimetallic materials, but to date its application has been limited to either modification of large area metal surfaces or nanoparticles in solution. Here, the localised surface modification of copper and silver substrates with gold through the galvanic replacement process is reported. This was achieved by generation of a localised flux of AuCl4− ions from a gold ultramicroelectrode tip which interacts with the unbiased substrate of interest. The extent of modification with gold can be controlled through the tip–substrate distance and electrolysis time.

Voltammetric monitoring of gold nanoparticle formation facilitated by glycyl-L-tyrosine : relation to electronic spectra and transmission electron microscopy images

Voltammetric techniques have been introduced to monitor the formation of gold nanoparticles produced via the reaction of the amino acid glycyl-L-tyrosine with Au(III) (bromoaurate) in 0.05 M KOH conditions. The alkaline conditions facilitate amino acid binding to Au(III), inhibit the rate of reduction to Au(0), and provide an excellent supporting electrolyte for voltammetric studies. Data obtained revealed that a range of time-dependent gold solution species are involved in gold nanoparticle formation and that the order in which reagents are mixed is critical to the outcome. Concomitantly with voltammetric measurements, the properties of gold nanoparticles formed are probed by examination of electronic spectra in order to understand how the solution environment present during nanoparticle growth affects the final distribution of the nanoparticles. Images obtained by the ex situ transmission electron microscopy (TEM) technique enable the physical properties of the nanoparticles isolated in the solid state to be assessed. Use of this combination of in situ and ex situ techniques provides a versatile framework for elucidating the details of nanoparticle formation.

Monitoring cuprous ion transport by scanning electrochemical microscopy during the course of copper electrodeposition

Scanning electrochemical microscopy (SECM), in the substrate generation–tip collection (SG-TC) mode, has been used to detect the cuprous ion intermediate formed during the course of electrodeposition of Cu metal from aqueous solution. Addition of chloride is confirmed to strongly stabilize the ion in aqueous solution and enhance the rate of Cu electrodeposition. This SECM method in the SG-TC mode offers an alternative to the rotating ring disk electrode (RRDE) technique for in situ studies on the effect of plating bath additives in metal electrodeposition. An attractive feature of the SECM relative to the RRDE method is that it allows qualitative aspects of the electrodeposition process to be studied in close proximity to the substrate in a simple and direct fashion using an inexpensive probe, and without the need for forced convection.

Scanning electrochemical microscopy study of the solid--solid interconversion of TCNQ to phase I and phase II CuTCNQ

The reduction of 7,7,8,8-tetracyanoquinodimethane (TCNQ) crystals attached to a glassy carbon electrode in the presence of Cu2+(aq) to form CuTCNQ(s) has been investigated using scanning electrochemical microscopy in the substrate generation tip collection mode and shown to involve a generation of soluble TCNQ−(aq). The subsequent oxidation of CuTCNQ does not involve simple expulsion of Cu+ into solution but a soluble complex attributed to Cu2+TCNQ−(aq). Mechanistic insights relative to the electrochemical conversion of CuTCNQ phase I into phase II by repetitive cycling of potential and electrochemical formation of KTCNQ have also been established

Local stress-strain distribution and load transfer across cartilage matrix at micro-scale using combined microscopy-based finite element method

Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.

Non-contact laser-scanning confocal microscopy of the human cornea in vivo

PURPOSE To investigate the utility of using non-contact laser-scanning confocal microscopy (NC-LSCM), compared with the more conventional contact laser-scanning confocal microscopy (C-LSCM), for examining corneal substructures in vivo. METHODS An attempt was made to capture representative images from the tear film and all layers of the cornea of a healthy, 35 year old female, using both NC-LSCM and C-LSCM, on separate days. RESULTS Using NC-LSCM, good quality images were obtained of the tear film, stroma, and a section of endothelium, but the corneal depth of the images of these various substructures could not be ascertained. Using C-LSCM, good quality, full-field images were obtained of the epithelium, subbasal nerve plexus, stroma, and endothelium, and the corneal depth of each of the captured images could be ascertained. CONCLUSIONS NC-LSCM may find general use for clinical examination of the tear film, stroma and endothelium, with the caveat that the depth of stromal images cannot be determined when using this technique. This technique also facilitates image capture of oblique sections of multiple corneal layers. The inability to clearly and consistently image thin corneal substructures - such as the tear film, subbasal nerve plexus and endothelium - is a key limitation of NC-LSCM.

Colloidal drug probe : method development and validation for adhesion force measurement using atomic force microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between the drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from a dry powder inhaler (DPI) formulation. Initially model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-Ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres preattached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies

High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.

Corneal confocal microscopy detects early nerve regeneration in diabetic neuropathy after simultaneous pancreas and kidney transplantation

Diabetic neuropathy is associated with increased morbidity and mortality. To date, limited data in subjects with impaired glucose tolerance and diabetes demonstrate nerve fiber repair after intervention. This may reflect a lack of efficacy of the interventions but may also reflect difficulty of the tests currently deployed to adequately assess nerve fiber repair, particularly in short-term studies. Corneal confocal microscopy (CCM) represents a novel noninvasive means to quantify nerve fiber damage and repair. Fifteen type 1 diabetic patients undergoing simultaneous pancreas-kidney transplantation (SPK) underwent detailed assessment of neurologic deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy, corneal sensitivity, and CCM at baseline and at 6 and 12 months after successful SPK. At baseline, diabetic patients had a significant neuropathy compared with control subjects. After successful SPK there was no significant change in neurologic impairment, neurophysiology, QST, corneal sensitivity, and intraepidermal nerve fiber density (IENFD). However, CCM demonstrated significant improvements in corneal nerve fiber density, branch density, and length at 12 months. Normalization of glycemia after SPK shows no significant improvement in neuropathy assessed by the neurologic deficits, QST, electrophysiology, and IENFD. However, CCM shows a significant improvement in nerve morphology, providing a novel noninvasive means to establish early nerve repair that is missed by currently advocated assessment techniques.

In vivo confocal microscopy of the ocular surface : from bench to bedside

In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.

Repeatability of confocal microscopy measurements on the central and peripheral cornea

Purpose To assess confocal microscopy repeatability (ConfoScan3, Nidek, Italy) when assessing the morphology of the limbus, midperipheral and central cornea. Method The central, mid-peripheral and limbal cornea (temporal and nasal) of the right eye of 8 subjects were examined with a ConfoScan3 in two different visits, at least six months apart. Bland-Altman repeatability was measured for 29 parameters: basal cell density and size, anterior and posterior keratocyte densities (AKD/PKD), endothelial cell density, polymegethism, pleomorphism, mean area and sides of endothelial cells - in the five different corneal areas examined. Results As a percentage of the mean absolute values, repeatability of 0–10% was classified as “excellent”, between 10–30% as “acceptable” and over 30% as “poor”. Repeatability was excellent for 14% of parameters and acceptable for 52% of parameters. The number of endothelial cell sides in the central cornea demonstrated the best repeatability (2.0%) whilst mid-temporal PKD showed the poorest repeatability (53.7%). Conclusions Confocal microscopy is at least an adequately repeatablemethodof evaluating the various corneal layers at different locations. Our dataset supports the ongoing use of the technique in research and clinical practice.

Comparing skin biopsy with confocal microscopy : diagnostic yield of nerve fiber density

Background and aims: The assessment of intra-epidermal nerve fiber density (IENFD) in skin biopsies and corneal nerve fiber density (CNFD) using corneal confocal microscopy (CCM) provides promising techniques to detect small nerve fiber damage in patients with peripheral neuropathy. To help define the clinical utility of each of these techniques in patients with diabetic neuropathy we have assessed sensitivity and specificity of IENFD and CNFD in predicting the following: 1) diabetic polyneuropathy (DPN); 2) risk of foot ulceration (RFU); 3) initial small fiber neuropathy (iSFN); 4) severe small fiber neuropathy (sSFN)...