MRT Letter: High resolution SEM imaging of nano‐architecture of cured urea‐formaldehyde resin using plasma coating of osmium

Nanoarchitecture of cured urea‐formaldehyde (UF) resins was examined with a field‐emission scanning electron microscope (FE‐SEM) after coating samples with osmium, which is considered to produce particles of considerably smaller size compared to other metal coatings used in SEM studies. This method enabled comparison of the nanoarchitecture of UF resins of low (1.0) and high (1.6) formaldehyde/urea (F/U) mole ratios to be made, based on imaging of extremely small size particles as part of UF resin architecture, not described before. Imaging revealed presence of relatively large globular particles (148.084–703.983 nm size range) as well as smaller substructures (28.004–39.604 nm size range) as part of the architecture of 1.0‐mole UF resin. Globular particles were also present in 1.6 mole UF resin, but of considerably smaller size (14.760–50.269 nm). The work presented demonstrates usefulness of osmium coating in unraveling the intricacies of the nanostructural organization of cured UF resins, prompting wider application of this immensely useful but grossly underutilized metal coating type in high resolution SEM examination of biological and materials samples. Microsc. Res. Tech. 76:1108–1111, 2013. © 2013 Wiley Periodicals, Inc.     

The innovative safe fixative for histology,histopathology, and immunohistochemistry techniques: “Pilot study using shellac alcoholic solution fixative”

The concerns over health and workplace hazards of formalin fixative, joined to its cross‐linking of molecular groups that results in suboptimal immunohistochemistry, led us to search for an innovative safe fixative. Shellac is a natural material which is used as a preservative in foods and pharmaceutical industries. This study was undertaken to evaluate the fixation adequacy and staining quality of histopathological specimens fixed in the “shellac alcoholic solution” (SAS), and also to determine the validity of immunohistochemical staining of SAS‐fixed material in comparison to those fixed in formalin. Fresh samples from 26 cases from various human tissues were collected at the frozen section room of King Abdulaziz University Hospital, and fixed in SAS fixative or in neutral buffered formaldehyde (NBF) for 12, 18, 24, and 48 h, and processed for paraffin sectioning. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and immunostained for different antigens. The tissues fixed in SAS for >18 h showed best staining quality of H&E comparable to NBF‐fixed tissues. Comparison of the immunohistochemical staining of different tissues yielded nearly equivalent readings with good positive nuclear staining quality in both fixatives. These findings support the fixation and preservation adequacy of SAS. Furthermore, it was concluded that the good staining quality obtained with SAS‐fixed tissues, which was more or less comparable with the quality obtained with the formalin fixed tissues, supports the validity of this new solution as a good innovative fixative. Microsc. Res. Tech. 77:385–393, 2014. © 2014 Wiley Periodicals, Inc.     

Poly(l‐lysine)‐graft‐poly(ethylene glycol): a versatile aqueous lubricant additive for tribosystems involving thermoplastics

The adsorption and aqueous lubricating behaviour of poly(l ‐lysine)‐graft‐poly(ethylene glycol) (PLL‐g‐PEG) have been investigated for tribopairs involving thermoplastic materials, including polypropylene, polyamide‐6,6 and polyethylene. A major finding is that PLL‐g‐PEG adsorbs onto both hydrophobic, non‐polar surfaces and hydrophilic, polar (negatively charged) surfaces from aqueous solution, and thus plays as a very unique and effective aqueous boundary lubricant additive for the sliding contact of thermoplastics against themselves as well as against many hydrophilic, polar materials, including metals (e.g. stainless steel) or ceramics (e.g. zirconia, ZrO₂). Copyright © 2007 John Wiley & Sons, Ltd.     

The effects of osmium tetroxide post‐fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO₄) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post‐fixed with 4% OsO₄, while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post‐fixation with 4% OsO₄ could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO₄ post‐fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.     

Synchrotron radiation X‐ray micro‐fluorescence: Protocol to study mesenchymal stem cells

The micro‐X‐ray fluorescence by synchrotron radiation (μ‐XRF) is a method to determine the composition of tissues without destroying the samples. However, this technique has never been used for the analysis of mesenchymal stem cells (MSC). This study compared different protocols for fixing, storing, preserving, and establishing the correct numbers of dental derived MSC submitted to μ‐XRF analysis. Stem cells were obtained from human dental tissue. After cell expansion, and MACS isolation, the samples were fixed and the following quantities of cells 1 × 10⁴ to 1 × 10⁷ were divided in two groups: G1: fixed in 4% paraformaldehyde diluted in phosphate‐buffered saline solution, and G2: fixed in 4% paraformaldehyde diluted in MilliQ water. The G1 cells showed precipitation of chemical components from the solution resulting in the formation of salt crystals while G2 cells were clear and almost transparent in the sample holder. With regards to cells concentration, the best results occurred when four droplets of 1 × 10⁷ cells were analyzed. This work shows that to identify and study the distribution of trace elements in MSC by μ‐XRF, the best protocol is fixation in 4% paraformaldehyde diluted with MilliQ water at 4°C and a concentration of four incremental droplets of 1 × 10⁷ cells. Microsc. Res. Tech. 79:149–154, 2016. © 2016 Wiley Periodicals, Inc.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

Morphological changes of poly(DI‐lactide‐co‐glycolide) nano‐particles containing ascorbic acid during in vitro degradation process

Poly(dl ‐lactide‐co‐glycolide) powder composed of uniform particles with the mean particle size in the range of 110–170 nm was obtained from commercial granules. Ascorbic acid in different concentrations was encapsulated into the poly(dl ‐lactide‐co‐glycolide) particles. Degradation of the latter in terms of morphological changes in the physiological solution was followed. Within a period of 2 months, the particles completely degrade and all the ascorbic acid is released. The samples were characterized by ultraviolet spectroscopy and scanning electron microscopy.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. I. Glutaraldehyde, osmium tetroxide, Araldite methods

The loss of ¹⁴C ethanolamine- and ³H choline-labelled phospholipids from rat liver during tissue preparation for electron microscopy has been examined. Column and thin-layer chromatography combined with double-label scintillation spectrometry were used to analyse the radioactive phospholipid content of the livers of rats injected simultaneously with ¹⁴C aminoethanol and ³H choline chloride. After 4 h (in vivo) the ¹⁴C and ³H labels were mainly incorporated into phosphatidyl ethanolamine and phosphatidyl choline respectively but some ¹⁴C label had been incorporated into phosphatidyl choline. Chopped rat liver was fixed in glutaraldehyde or osmium tetroxide or both sequentially and tissues were dehydrated in ethanol and embedded in Araldite. In each procedure examined the choline label proved more labile than the ethanolamine. After glutaraldehyde fixation alone complete loss of phosphatidyl choline occurred and half of the phosphatidyl ethanolamine was also lost. Following osmium tetroxide fixation negligible loss of either phosphatide occurred. In terms of phospholipid retention, no advantage was gained by glutaraldehyde fixation prior to osmium tetroxide fixation. The results show that both ethanols and embedding monomers are potent phospholipid solvents. The data also suggests that EM autoradiography of these two phosphatides may be carried out with reasonable confidence although it must be pointed out that a high degree of retention does not necessarily imply retention in situ.     

Label‐free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

We introduce a label‐free spectroscopic method to classify subtypes of quinolone‐nonsusceptible Escherichia coli (E. coli ) isolates obtained from human blood cultures. Raman spectroscopy with a 30‐nm gold‐deposited, surface‐enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)‐predefined groups including E . coli ATCC25922, E . coli ST131:O75, and E . coli ST1193:O25b. Although there was a coffee‐ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001–1004 cm^?1 (C? C aromatic ring breathing stretching vibrational mode of phenylalanine), 1447–1448 cm^?1 (C? H₂ scissoring deformation vibrational mode), and 1667 cm^?1 (amide I α‐helix). Although the three MLST‐predefined E . coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm‐assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone‐nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone‐nonsusceptible E . coli ST131:O75 and E . coli ST1193:O25b isolates. Therefore, this label‐free and nondestructive technique is likely to be useful for clinically diagnosing quinolone‐nonsusceptible E. coli isolates with the MLST method.     

Investigation of properties of electrochemically synthesizediron oxide nano‐powders

Nano‐sized powders of iron oxides have been synthesized electrochemically at temperatures in the range of 295–361 K, and current densities in the range of 200–1000 mA dm^?2. The structure and morphology of the powders were investigated by X‐ray diffraction and scanning electron and transmission electron microscopy techniques. Their infrared absorption spectra, specific heat C_p(T) and magnetic susceptibility χ(T) temperature dependences are also determined. The obtained powders consist of two phases, each possessing distinguished characteristics: the one formed of large plates and the other of whiskers. By appropriate adjustment of the synthesis conditions, it is possible to change features and relative abundances of the two phases, and that way to control morphology and other powder properties. Relaxation and transformation of the phases under external influences was also investigated, and the optimal procedure for preparation and stabilization of iron oxide nano‐sized powders with desired characteristics was established.     

Anatomical analysis of turgescent and semi‐dry resurrection plants: The effect of sample preparation on the sample,resolution, and image quality of X‐ray micro‐computed tomography (μCT)

Computer tomography has been used frequently for the 3‐D visualization of plant anatomical traits but sample preparation has been widely neglected. Without any preparation smaller (i.e., up to 1 × 1 cm²) turgescent or semi‐dry plant samples (especially leaf samples) diminish the image quality of a scan due to gradual water loss and therefore constant movement. A suitable preparation for scans of turgescent and semi‐dry plant samples with a high resolution μCT (<1–5 μm) has to be very thin, heat‐resistant (up to 35°C), have a low attenuation coefficient, and should not alter the water content and structure of the sample. Several agents have been tested, but only a coating with vaseline conserved the water content of a plant sample efficiently. However, water molecules and vaseline both attenuate the X‐ray beam, which decreases the image quality of scans of turgescent or semi‐dry plant samples. Therefore, trade‐offs between the spatial resolution, sample water content, sample size, and image quality have to be considered: larger samples have to be placed further away from the X‐ray tube, which leads to a lower spatial resolution; water and preparation agents attenuate the X‐ray beam, causing low‐quality images which may be accompanied by motion artifacts compared to a scan of a dry sample, where no preparation is necessary. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Feasibility of high pressure freezing with freeze substitution after long‐term storage in chemical fixatives

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non‐cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols. Microsc. Res. Tech. 76:942–946, 2013. © 2013 Wiley Periodicals, Inc.     

An innovative,interdisciplinary, and multi‐technique study of gilding and painting techniques in the decoration of the main altarpiece of Miranda do Douro Cathedral (XVII‐XVIIIth centuries,Portugal)

The research results presented in this paper are part of a larger study on the materials and techniques used in polychrome altarpieces of gilded woodcarving decoration (“talha dourada”) in Portugal. The paper focuses on a narrative Portuguese Altarpiece from Miranda do Douro, considered one of the masterpieces of “talha dourada” among all the retables of the Iberian Peninsula in XVII^th and XVIII^th centuries. Although on the Portuguese territory, the altarpiece was made by artists from the Royal Spanish school of Valladolid, under a mannerist style. Thus the study opens a window on the artists' circulation between Spain and Portugal and influences of the Spanish schools in Baroque epoch on the Portuguese “talha”. During its history this altarpiece underwent several transformations and extensive conservation treatments in 1989. On this occasion more than 50 samples were collected and analyzed using an interdisciplinary multi‐technique methodology. 27 of these samples are chosen for this study in order to investigate the chromatic palette, the materials and techniques used in the polychromy of the retable. A novel protocol of investigation using different conventional and unconventional analytical techniques (OM + fluorescent staining tests on cross‐sections, Raman microscopy, XRD, XRF, X‐ray micro‐CT, SEM‐EDX, MALDI‐TOF‐MS and LC‐MS/MS) was established within an innovative research project ( http://sites.fct.unl.pt/gilt‐teller/ ) and applied on these samples. This protocol is necessary to confirm the results obtained in the 1989 campaign and to have further insight into the gilding and polychrome decoration materials and techniques and the additional information reported in the historical documents. The material and technical history of this important altarpiece will be thus re‐documented from a scientific perspective, meant to confirm and bring new information on the decorative technique used in the creation of this complex Portuguese monument. Microsc. Res. Tech. 76:733–743, 2013. © 2013 Wiley Periodicals, Inc.     

Quantification of anhydrous ethanol and detection of adulterants in commercial Brazilian gasoline by Raman spectroscopy

Raman spectroscopy can be used to evaluate the quality of fuels in a remote, rapid, and nondestructive manner without the need for reagents. In this study, Raman was used to quantify anhydrous ethanol in commercial gasoline and to detect peaks due to compounds commonly used for the adulteration of commercial gasoline. Samples of commercial gasoline were collected from fuel stations in the region of Santos, SP, Brazil. Samples of naphtha from the refinery, pure ethanol, and ethanol diluted in distilled water at concentrations close to the range used in the gasoline were also obtained and characterized. Raman spectra were collected using a dispersive Raman spectrometer (830?nm, 2?cm^?1 resolution in the 400–1800?cm^?1 spectral range). As expected, the spectra of commercial gasoline showed pronounced peaks of naphtha and ethanol. By using the peak intensities of the ethanol diluted in water, the ethanol concentration was found to be in the range of 27%?±?1% in most of the samples; some samples presented ethanol concentrations as high as 28.8%, suggesting adulteration. Some samples presented peaks at 766, 798, and 995?cm^?1 with higher intensities, suggesting the presence of an adulterant with organic characteristics, such as solvents with aromatic rings. Raman spectroscopy has been shown to be effective in determining the adulteration of commercial gasoline, which may contribute to rapid quality control of fuels at the point of sale.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell-substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell–substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

Effect of heat treatment on the microstructure and properties of Ni based soft magnetic alloy

A Ni‐based alloy was heat treated by changing the temperature and ambient atmosphere of the heat treatment. Morphology, crystal structure, and physical performance of the Ni‐based alloy were characterized via SEM, XRD, TEM, and PPMS. Results show that due to the heat treatment process, the grain growth of the Ni‐based alloy and the removal of impurities and defects are promoted. Both the orientation and stress caused by rolling are reduced. The permeability and saturation magnetization of the alloy are improved. The hysteresis loss and coercivity are decreased. Higher heat treatment temperature leads to increased improvement of permeability and saturation magnetization. Heat treatment in hydrogen is more conducive to the removal of impurities. At the same temperature, the magnetic performance of the heat‐treated alloy in hydrogen is better than that of an alloy with heat treatment in vacuum. The Ni‐based alloy shows an excellent magnetic performance on 1,373 K heat treatment in hydrogen atmosphere. In this process, the µ_m, B_s, P_u, and H_c of the obtained alloy are 427 mHm^?1, 509 mT, 0.866 Jm^?3, and 0.514 Am^?1, respectively. At the same time, the resistivity of alloy decreases and its thermal conductivity increases in response to heat treatment.     

Nitric oxide regulates mitochondrial re‐modelling in interscapular brown adipose tissue: ultrastructural and morphometric‐stereologic studies

As a complex, cell‐specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of l ‐arginine‐nitric oxide (NO)‐producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving l ‐arginine, a substrate of NO synthases (NOSs), or N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub‐groups – acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. l ‐Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold‐induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in l ‐arginine‐treated rats. Ultrastructural examinations of l ‐arginine‐treated cold‐acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well‐ordered cristae in their profiles), which however were inactive in l ‐arginine‐receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, l ‐NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.     

Near-field and confocal surface-enhanced resonance Raman spectroscopy at cryogenic temperatures

For laser spectroscopy at variable temperatures with high spatial resolution a combined scanning near‐field optical and confocal microscope was developed. Rhodamine 6G (R6G) dye molecules dispersed on silver nano‐particles or nano‐clusters were investigated. For optical excitation of the molecules, either an aperture probe or a focused laser spot in confocal arrangement were employed. Raman spectra in the wavenumber range between 300 cm^?1 and 3000 cm^?1 at room temperatures down to 8.5 K were recorded. Many of the observed Raman lines can be associated with the structure of the adsorbed molecule. Intensity fluctuations in spectral sequences were observed down to 77 K and are indicative of single molecule sensitivity.     

Optimizing use of the structural chemical analyser (variable pressure FESEM‐EDX raman spectroscopy) on micro‐size complex historical paintings characterization

The novel Structural Chemical Analyser (hyphenated Raman spectroscopy and scanning electron microscopy equipped with an X‐ray detector) is gaining popularity since it allows 3‐D morphological studies and elemental, molecular, structural and electronic analyses of a single complex micro‐sized sample without transfer between instruments. However, its full potential remains unexploited in painting heritage where simultaneous identification of inorganic and organic materials in paintings is critically yet unresolved. Despite benefits and drawbacks shown in literature, new challenges have to be faced analysing multifaceted paint specimens. SEM?Structural Chemical Analyser systems differ since they are fabricated ad hoc by request. As configuration influences the procedure to optimize analyses, likewise analytical protocols have to be designed ad hoc. This paper deals with the optimization of the analytical procedure of a Variable Pressure Field Emission scanning electron microscopy equipped with an X‐ray detector Raman spectroscopy system to analyse historical paint samples. We address essential parameters, technical challenges and limitations raised from analysing paint stratigraphies, archaeological samples and loose pigments. We show that accurate data interpretation requires comprehensive knowledge of factors affecting Raman spectra. We tackled: (i) the in‐FESEM?Raman spectroscopy analytical sequence, (ii) correlations between FESEM and Structural Chemical Analyser/laser analytical position, (iii) Raman signal intensity under different VP‐FESEM vacuum modes, (iv) carbon deposition on samples under FESEM low‐vacuum mode, (v) crystal nature and morphology, (vi) depth of focus and (vii) surface‐enhanced Raman scattering effect. We recommend careful planning of analysis strategies prior to research which, although time consuming, guarantees reliable results. The ultimate goal of this paper is to help to guide future users of a FESEM‐Structural Chemical Analyser system in order to increase applications.