Visualisation of biopolymer mixtures using confocal scanning laser microscopy (CSLM) and covalent labelling techniques

Confocal scanning laser microscopy (CSLM) has been used to study the behaviour of mixtures of proteins, gelatine, whey proteins and β-lactoglobulin, and polysaccharides, dextran, gellan gum, carrageenan, gum Arabic, and starch. CSLM proved to be a suitable technique to visualise the microstructure of these (phase separated) mixtures in two and three-dimensional images. Contrast through fluorescence is obtained either by covalent labelling (polysaccharides and proteins) or non-covalent labelling (proteins and starch). Double and triple labelling allows the visualisation of individual components in a complex mixture of biopolymers.     

Fluorescence staining and confocal laser scanning microscopy study of hydrogen-bonded poly(vinylpyrrolidone)/poly(acrylic acid) film

Poly(vinylpyrrolidone) (PVPON) and poly(acrylic acid) (PAA) were layer-by-layer (LBL) assembled to prepare the thin films based on hydrogen-bonding complexation. The hydrogen-bonded PVPON/PAA films were incubated in acidic, neutral and basic vapors separately. To study the morphologies after incubation, the films were stained by pH-sensitive fluorescent dyes using chemical and physical ways, and investigated with confocal laser scanning microscope (CLSM). The chemical way (labeling) was covalently linking fluoresceinamine (FAM) to some monomer units of PAA while the physical way was adsorbing rhodamine B (RB) molecules from dilute solution. Atomic force microscope (AFM) was combined with CLSM to find that after incubation in neutral or basic vapor the hydrogen-bonded PVPON/PAA films form porous structure and the pores are through the whole film.     

Effect of sucrose on molecular and interaction parameters of sodium caseinate in aqueous solution: relationship to protein gelation

The effect of sucrose on molecular and interaction parameters of sodium caseinate in aqueous medium has been investigated using static and dynamic multi-angle laser light scattering over a wide range of sucrose concentration (from 10 to 78 w/v%) and pH values (from 7.0 to 3.5). Measurements have been made of the molar mass, the radius of gyration, the hydrodynamic radius, and the second virial coefficient of sodium caseinate in aqueous solution. Pronounced dissociation of sodium caseinate sub-micelles¹ was found in the presence of sucrose at a pH above the protein's isoelectric point. The effect of sucrose at a pH near the isoelectric point is very different. This is reflected in the pronounced increase in molar mass, radius of gyration, and the difference between the radius of gyration and the hydrodynamic radius. It was found that the extent of the protein association, caused by the presence of sucrose, is a key factor contributing to the hydrophobic–hydrophilic balance of the protein surface, and hence to the thermodynamic affinity of the caseinate sub-micelles for the aqueous medium and for each other. Analysis of light-scattering data using structure-sensitive plots shows a clear transition from Gaussian to wormlike chain/rod behaviour for sodium caseinate on pH lowering. Apparent relationships between the effects of sucrose on the self-association of sodium caseinate and a marked enhancement of the viscoelasticity of acid-induced casein gels have been revealed. Moreover, the dissociation of sodium caseinate sub-micelles is in excellent agreement with the more homogeneous microstructure of acid-induced protein gels in the presence of sucrose as detected by confocal laser scanning microscopy. We discuss likely molecular mechanisms underlying the observed effects of sucrose on the interactions and rheology in acidified caseinate systems.     

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     

Detailed analysis of membrane adsorber pore structure and protein binding by advanced microscopy

Commercial Sartobind^® porous cation exchanger membranes, based on stabilized regenerated cellulose and with sulfonic acid (S) or carboxylic acid groups (C), were analysed with respect to their pore structure in dry, slightly swollen and wet state by three microscopic methods, conventional scanning electron microscopy (SEM), environmental SEM (ESEM), and confocal laser scanning microscopy (CLSM). The dehydration behaviour of the membranes was in situ observed at varied vapour pressure in the chamber of the ESEM, indicating some deformations of the macropore structure (largest pore diameters up to 20 μm) and significant changes in dimension and mobility of smaller cellulose fibers within these macropores, both as function of water content of the membrane. The binding of mono-Cy5-labelled lysozyme inside fluoresceine-labelled and unlabelled Sartobind^® membranes was monitored by CLSM. The characteristic fluorescence intensity distributions in areas of (146 μm × 146 μm) indicated that protein binding takes place predominately in a layer which is anchored to a fine cellulose fiber network and, to a lower degree, directly to thick cellulose fibers. Due to the limited thickness of this binding layer, a significant fraction of the macropores remained free of protein. Protein binding as function of concentration and incubation times was also monitored by CLSM and discussed related to the binding isotherms for the membrane Sartobind^® S and C. Further, a flow-through cell for the in situ monitoring with CLSM of protein binding during the binding step was built, and the results obtained for binding of lysozyme in membranes Sartobind^® S indicate this experiment can give very important information on the dynamic behaviour of porous membrane adsorbers during separation: the lateral microscopic resolution in the x, y plane enables the identification of different breakthrough times as function of the location (pore structure), and this information can help to explain possible reasons for axial dispersion (in z-direction) observed in breakthrough analyses of the same separation in a chromatography system. The combination of advanced microscopy with detailed investigations of static and dynamic protein binding will provide a better understanding of the coupling between mass transfer and reversible binding in membrane adsorbers onto separation performance, and it will provide valuable guide-lines for the development of improved membrane adsorbers.     

Evaluation of hydrogen ion concentrations in prostates from rats and dogs using fluorescent confocal microscopy

The knowledge of intracellular spatial distribution of pH in prostates in animal models reflective of human prostate may have implications for drug development upon pH dependent drug delivery and activity. Freshly dissected prostate tissues (in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes and viewed using confocal microscopy. Images were initially taken in tissues perfused with RPMI-1640 medium. Calibration in situ was performed with high potassium buffers of known pH containing nigericin. Acetoxymethyl ester carboxy-SNARF-1 was visible in epithelial cells (but not stroma) in rat and dog prostates. The pH of lysosomes in prostate epithelial cells was 5.2 as determined by fluorescence of Lyso Sensor Green DND-189. A method of in situ confirmation of tissue viability was developed by a secondary loading and visualization of the BCECF fluorescent dye. Besides the direct measurement of the pH in rat and dog tissues (pH ≈ 7.0), a method of pH measurement in prostate tissue (rather than in cell culture) was developed.     

Interplay between electrochemistry and optical imaging: The whole is greater than the sum of the parts

Optical techniques can afford a powerful characterisation of the solid–liquid interface that is composed of an electrode immersed in an electrolyte. While a typical electrochemical measurement such as current intensity is averaged over the entire electrode surface, the access to surface heterogeneity can provide an increased level of information enabling to rationalise and optimise the performance of chemical or biochemical sensors. In this opinion article, we will briefly review the different strategies developed to translate an electrochemical process into a luminescence signal. Also, several key examples will be selected and commented in order to highlight the key advantages of coupling electrochemistry with optical imaging, essentially fluorescence and electrochemiluminescence.     

Internalization of PLGA nanoparticles coated with poloxamer 188 in glioma cells: A confocal laser scanning microscopy study

Internalization of poloxamer 188-coated PLGA nanoparticles (NPs) in GL261 murine glioma cells was studied using confocal laser scanning microscopy. For visualization, both poloxamer 188 (P188) and PLGA were labeled covalently with fluorescent dyes Rhodamine B and Cyanine5, respectively. The results indicated that the PLGA NPs coated with poloxamer 188 enter a cell as an integral core–shell structure, which can be helpful for gaining further insight into the in vivo performance of surfactant-coated polymeric NPs as core–shell delivery systems     

Activated sludge characterization through microscopy: A review on quantitative image analysis and chemometric techniques

In wastewater treatment processes, and particularly in activated sludge systems, efficiency is quite dependent on the operating conditions, and a number of problems may arise due to sludge structure and proliferation of specific microorganisms. In fact, bacterial communities and protozoa identification by microscopy inspection is already routinely employed in a considerable number of cases. Furthermore, quantitative image analysis techniques have been increasingly used throughout the years for the assessment of aggregates and filamentous bacteria properties. These procedures are able to provide an ever growing amount of data for wastewater treatment processes in which chemometric techniques can be a valuable tool. However, the determination of microbial communities’ properties remains a current challenge in spite of the great diversity of microscopy techniques applied. In this review, activated sludge characterization is discussed highlighting the aggregates structure and filamentous bacteria determination by image analysis on bright-field, phase-contrast, and fluorescence microscopy. An in-depth analysis is performed to summarize the many new findings that have been obtained, and future developments for these biological processes are further discussed.     

Investigation of copper patinas using ion beam analysis and scanning electron microscopy

Ion beam analysis (IBA) techniques were applied successfully to the investigation of non‐corroded and artificially corroded patina layers grown on copper substrates in order to explore their potential use in the study of degradation phenomena of copper and copper alloys subjected to chemical treatment and exposed to selected environmental conditions. Rutherford backscattering spectroscopy (RBS) with deuterons as projectiles and the nuclear reactions ¹⁶O(d,p)¹⁷O and ³²S(p,p′γ)³²S were applied to the investigation of the depth distribution of oxygen and sulphur in near‐surface layers of synthetic patina consisting of mineral phases corresponding to chalcanthite as well as to cuprite + chalcanthite and antlerite + brochantite + chalcanthite. Electrochemical techniques (potentiodynamic polarization and cyclic voltammetry in 0.5 M Na₂SO₄) were used for artificial acceleration and study of the corrosion processes, and scanning electron microscopy (SEM/EDS) was used for examination of the surface morphology of the samples. A patinated roof sample from the Vienna Hofburg also was investigated using the same techniques. The measurement showed that IBA can provide valuable information for the study of patina near‐surface layers of thickness up to a few micrometres and indicated that cuprite was the mineral phase primarily formed on the copper substrates and the main component of the interface between the patina layer and the metallic substrate. The investigated copper patinas looked rather heterogeneous and were characterized by high porosity. Mixed patinas exhibited considerable stability to further corrosive attack. Copyright © 2005 John Wiley & Sons, Ltd.     

Optimization of the parameters for nickel electrowinning using interference microscopy and digital image analysis

Ni electrodeposition experiments at a constant current density 220 A/m² were performed to determine the optimum concentrations of chloride and an organic additive used for an industrial Ni electrowinning. White light interference microscopy was used to acquire digital images of the morphology of the electrodeposited nickel. The scaling analysis was employed to parameterize the morphological information encoded in the images. The standard deviation of the surface height, δ, the critical scaling length, L _c, and the optical roughness, 4δ/L _c, were determined as a function of the chloride concentration and the amount of organic additives. These parameters were plotted as a function of the two compositional variables. These three-dimensional plots allowed us to find conditions corresponding to the minimum of 4δ/L _c, at which the deposited nickel is well leveled. Dedicated to Prof O.A. Petrii on the occasion of his 70th birthday and in recognition of his contribution to electrochemistry.     

Surface orientation characterisation of rough mc‐silicon surfaces by confocal microscopy and EBSD

For many semiconductor and photovoltaic applications the quality of substrate surfaces is an important requirement for the development of new devices. Therefore, methods are needed to analyse the surface structure in detail. In the following a new method is described that allows a fast analysis of as‐cut or artificially modified and textured surfaces. It uses laser confocal microscopy images. Therefrom the orientation of small surface elements are detected and depicted in an orientation distribution function of surface normals. The method is applied to as‐sawn mc‐silicon surfaces. It will be shown that it is also possible to determine the grain orientation for grains near 〈111〉 crystal direction by this method. The combination of electron backscatter diffraction and orientation distribution function analysis enables the detailed investigation of surface elements of an anisotropic etched silicon wafer. Inhomogeneity of surface textures can easily and rapidly be visualised by this method. Copyright © 2012 John Wiley & Sons, Ltd.     

Archaeopolymetallurgical study of materials from an Iberian culture site in Spain by scanning electron microscopy with X-ray microanalysis, chemometrics and image analysis

Archaeometallurgical materials from “La Bastida de Moixent”, a site in Valencia (Spain), from the second Iberian iron age (4th Century B.C.) have been studied using metallographic techniques, microanalysis, chemometrics and image analysis. The materials come from various phases of iron production and cupellation of argentiferous lead to obtain silver. Scanning electron microscopy (SEM) is used to determine the morphological, microstructural and topographic characteristics of the samples. Image analysis was used to obtain a numeric estimate of the main components in these materials. X-ray microanalysis (SEM/EDX) provides qualitative and quantitative information about the elements in the sample. The semiquantitative results have been treated by chemometric tools such as Principal Component Analysis (PCA) to group the different archaeological materials. These analyses provide information on the lead and iron metallurgical processes, such as silver production by cupellation used in the period. Macroscopic and microscopic analyses indicate a high quality, acceptably uniform manufactured product, indicating broad technical skill in the later metallurgical process of transformation and refinement of these materials to obtain ingots and manufactured products to trade.     

Confocal microscopy study of uptake kinetics of α‐lactalbumin and β‐lactoglobulin onto the cation‐exchanger SP Sepharose FF

Confocal laser scanning microscopy (CLSM) was used to study single‐ and two‐component protein uptake for α‐lactalbumin (ALA) and β‐lactoglobulin (BLG), as models for whey proteins, to SP Sepharose FF at pH 3.7 during batch experiments in a finite bath. By coupling a fluorescent dye with the protein molecule, the penetration into individual adsorbent particles at different times during batch uptake was visualised. In a single‐component system, BLG penetrated fast into the adsorbent beads and gradually filled them in a shell‐wise fashion, while adsorption of ALA was mostly confined to the outer shells of the adsorbent. For the two‐component studies, the results showed that ALA was able to displace BLG despite its lower affinity to the adsorbent under the employed conditions. CLSM results were then compared both qualitatively and quantitatively to their counterparts obtained in traditional experiments by indirect measurements of the protein concentration in the fluid phase. A novel quantitative approach was undertaken by modifying the simple kinetic rate model traditionally used to determine the kinetic rate constant, k₁, for batch uptake experiments, in order to describe batch uptake kinetics based on CLSM data. Although BLG results were in good agreement, there was a discrepancy in ALA results.     

Multispectral and hyperspectral image analysis of elemental and micro-Raman maps of cross-sections from a 16th century painting

Spectroscopic imaging is well suited to the study of micro-samples from artworks, where the sample material is limited and the maximum amount of information needs to be obtained. In this study, a new approach to imaging elemental data from energy dispersive X-ray analysis maps was used in conjunction with micro-Raman spectroscopic imaging to characterise the paint layers within micro-samples. Cross-sections from the 16th century painting Portrait of a Youth were found to contain vermilion, lead-tin yellow type 1 and a blue-green pigment consistent with terre-verte. The mid-preparatory layer (imprimatura) contains a high proportion of elements and mineral inclusions that indicates a clay-type composition. The ground layer was identified as anhydrite with large gypsum inclusions. The pigments and composition of the preparatory layers are consistent with those used by Italian Renaissance artist Dosso Dossi.     

Vector potential photoelectron microscopy: hyperspectral image processing and super‐resolution applied to images of a Ca–Al alloy intermetallic phase distribution

Vector potential photoelectron microscopy (VPPEM) produces four dimensional hyper‐spectral data, and image processing is an integral part of the experimental technique. VPPEM is a new class of instrument, and this is the first discussion of some of the data reduction techniques that have been found effective. The point spread function of VPPEM is multidimensional with a high‐frequency component. Although this high‐frequency component is a small fraction of the total spatial response, images with good signal‐to‐noise can be spatially deconvolved to give super resolution images. The VPPEM data reduction process is illustrated by the analysis of the multiple intermetallic phases in a Ca–Al alloy. These phases have been imaged with better than 0.5‐μ spatial resolution. Not all the problems with the data reduction process have been satisfactorily dealt with, and lessons from this work will influence the design of future instruments. Copyright © 2014 John Wiley & Sons, Ltd.     

Scanning electron microscopy and energy dispersive analysis: applications in the field of cultural heritage

Scanning electron microscopy has been extensively used for the material characterization of objects of artistic and archaeological importance, especially in combination with energy dispersive X-ray microanalysis (SEM/EDX). The advantages and limitations of SEM/EDX are presented in a few case studies: analysis of pigments in cross-sections of paint layers, quantitative analysis of archaeological glass from the Roman period excavated in Ephesos/Turkey, and investigations on glasses with medieval composition concerning their weathering stability and degradation phenomena.     

Profiling analysis of volatile compounds from fruits using comprehensive two-dimensional gas chromatography and image processing techniques

An image processing approach originating from the proteomics field has been transferred successfully to the processing of data obtained with comprehensive two-dimensional gas chromatographic separations data. The approach described here has proven to be a useful analytical tool for unbiased pattern comparison or profiling analyses, as demonstrated with the differentiation of volatile patterns (“aroma”) from fruits such as apples, pears, and quince fruit. These volatile patterns were generated by headspace solid phase microextraction coupled to comprehensive two-dimensional gas chromatography (HS-SPME-GC × GC). The data obtained from GC × GC chromatograms were used as contour plots which were then converted to gray-scale images and analyzed utilizing a workflow derived from 2D gel-based proteomics. Run-to-run variations between GC × GC chromatograms, respectively their contour plots, have been compensated by image warping. The GC × GC images were then merged into a fusion image yielding a defined and project-wide spot (peak) consensus pattern. Within detected spot boundaries of this consensus pattern, relative quantities of the volatiles from each GC × GC image have been calculated, resulting in more than 700 gap free volatile profiles over all samples. These profiles have been used for multivariate statistical analysis and allowed clustering of comparable sample origins and prediction of unknown samples. At present state of development, the advantage of using mass spectrometric detection can only be realized by data processing off-line from the identified software packages. However, such information provides a substantial basis for identification of statistically relevant compounds or for a targeted analysis.