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BACKGROUND: Developing disposable paper-based devices has positively impacted analytical science, particularly in developing countries. Some benefits of those devices include their versatility, affordability, environmentally friendly, and the possibility of being integrated with portable electrochemical or colorimetric detectors. Paper-based analytical devices (PADs) comprising circular zones and microfluidic networks have been successfully employed in the analytical chemistry reign. However, the combination of the stencil-printing method and alternative binder has not been satisfactorily explored for fabricating colorimetric paper devices. RESULTS: We developed PADs exploring the stencil printing approach and glass varnish as the hydrophobic chemical agent. As a proof-of-concept, the colorimetric assay of salivary α-amylase (sAA) was performed in saliva samples. Through the scanning electron microscopy measurements, it was possible to indicate satisfactory definitions between native fibers and barrier, and that the measured values for the channel width revealed suitable fidelity (R2 = 0.99) with the nominal widths (ranging from 400 to 5000 µm). The proposed hydrophobic barrier exhibited excellent chemical resistance. The analytical applicability for detecting sAA revealed linear behavior in the range from 2 to 12 U mL-1 (R2 = 0.99), limit of detection of 0.75 U mL-1, reproducibility (RSD ≤2.4%), recovery experiments ranged from 89 to 108% and AGREE response (0.86). In addition, the colorimetric analysis of sAA in four different saliva samples demonstrated levels ranging from 202 to 2080 U mL-1, which enabled monitoring the absence and presence of periodontitis. SIGNIFICANCE: This report has presented the first use of a self-adhesive mask and glass varnish for creating circular zones and microfluidic architectures on paper without using thermic or UV curing treatments. Also, the proposed analytical methodology for detecting sAA exhibited suitable ecological impact considering the AGREE tool. We believe the proposed fabrication of paper devices emerges as a novel, simple, high-fidelity microfluidic channel and portable analytical approach for colorimetric sensing.

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Bee pollen is considered an excellent dietary supplement with functional characteristics, and it has been employed in food and cosmetics formulations and in biomedical applications. Therefore, understanding its chemical composition, particularly crude protein contents, is essential to ensure its quality and industrial application. For the quantification of crude protein in bee pollen, this study explored the potential of combining digital image analysis and Random Forest algorithm for the development of a rapid, cost-effective, and environmentally friendly analytical methodology. Digital images of bee pollen samples (n = 244) were captured using a smartphone camera with controlled lighting. RGB channels intensities and color histograms were extracted using open source softwares. Crude protein contents were determined using the Kjeldahl method (reference) and in combination with RGB channels and color histograms data from digital images, they were used to generate a predictive model through the application of the Random Forest algorithm. The developed model exhibited good performance and predictive capability for crude protein analysis in bee pollen (R2 = 80.93 %; RMSE = 1.49 %; MAE = 1.26 %). Thus, the developed analytical methodology can be considered environmentally friendly according to the AGREE metric, making it an excellent alternative to conventional analysis methods. It avoids the use of toxic reagents and solvents, demonstrates energy efficiency, utilizes low-cost instrumentation, and it is robust and precise. These characteristics indicate its potential for easy implementation in routine analysis of crude protein in bee pollen samples in quality control laboratories.

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Tomato fruit is susceptible to chilling injury (CI) during its postharvest handling at low temperature. The symptoms caused by this physiological disorder have been commonly evaluated by visual inspection at a macro-observation scale on fruit surface; however, the structure at deeper scales is also affected by CI. This work aimed to propose a descriptive model of the CI development in tomato tissue under the micro-scale, micro-nano-scale and nano-scale approaches using fractal analysis. For that, quality and fractal parameters were determined. In this sense, light microscopy, Environmental Scanning Electron Microscopy (ESEM) and Atomic Force Microscopy (AFM) were applied to analyse micro-, micro-nano- and nano-scales, respectively. Results showed that the morphology of tomato tissue at the micro-scale level was properly described by the multifractal behaviour. Also, generalised fractal dimension (Dq=0) and texture fractal dimension (FD) of CI-damaged pericarp and cuticle were higher (1.659, 1.601 and 1.746, respectively) in comparison to non-chilled samples (1.606, 1.578 and 1.644, respectively); however, FD was unsuitable to detect morphological changes at the nano-scale. On the other hand, lacunarity represented an appropriate fractal parameter to detect CI symptoms at the nano-scale due to differences observed between damaged and regular ripe tissue (0.044 and 0.025, respectively). The proposed multi-scale approach could improve the understanding of CI as a complex disorder to the development of novel techniques to avoid this postharvest issue at different observation scales.

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This data article describes the connected pore cluster data from segmented nano-images of rocks related to a geothermal system. The collected samples include two (2) vesicle-amygdaloidal basalt (host rock) and four (4) horizons collected from a siliceous sinter mound (rock precipitated from hot waters). All the samples have undergone computed tomography scanning using a SkyScan 2211 multiscale X-ray nano-CT system (Bruker®), and the slices were analyzed using the Digital Rock Physics (DRP) approach. Pore volume and fluid permeability in the three directions were calculated with scripts of Python (v.3.9) and the visualizations of the 3D models were run with Paraview (v.5.10) software. The petrophysical properties, diagrams, and figures were produced by stacking the 2D projections (8-bit grayscale *.png images format) from the scanning. Raw data (images) were deposited in a repository, which has granted a persistent identifier (Mendeley Data: https://data.mendeley.com/datasets/srpxhpd37p/2). This article provides a study case to handle the data that test the interconnectivity and ability to transport fluids and/or exogenous matter carried during high-flow events in rocks outcropping at the surface level of a geothermal system.

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Pyknosis or hypercondensation of chromatin is informative in the understanding of nucleosomal packing in translationally inactive chromatin and in the compression of cell death. However, mechanisms that result in the formation of avian erythrocytes with variant nuclear morphology are poorly understood.Purpose: In this work, we evaluated pyknosis in pigeon erythrocytes treated with thermal stress using Digital Image Analysis (DIA).Materials and methods: Pigeon erythrocytes were treated at thermal stress (33 °C, 43 °C, and 53 °C), and nuclear modifications were analyzed by DIA.Results: Our results showed that thermal stress induced DNA condensation. Based on DNA fluorescent staining and compaction, four subclasses with progressively more pyknotic nuclei each could be distinguished. Alkaline comet assay showed that the presence of pyknotic nuclei was associated with the DNA fragmentation typical of apoptosis. DIA analysis showed a decrease of nuclear area and a significant increase of fluorescence intensity with respect to non-pyknotic nucleus. Additionally we observed nuclear dissolution events associated with swell and loose membrane integrity.Conclusion: These findings can contribute to the evaluation of health and metabolic status in diagnostic cytology, especially in neoplastic conditions and infection by microorganisms.

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In this work, we describe for the first time the synthesis of a thiocarbazone for the selective determination of Cu2+ in distilled beverages. The method was based on the complexation reaction of Cu2+ with the thiocarbazone, and the colored product was analyzed using a smartphone application. The thiocarbazone reacts with Cu2+ to form a 1:1 (metal:ligand) complex. The Cu2+ complex was characterized by UV, IR and NMR spectral analyses. The proposed reaction yields a yellow color, and therefore, channel B of the RGB system was used in the analysis. After optimizing the reaction conditions, an analytical curve was obtained to determine Cu2+ concentrations ranging between 0.25 and 6.75 mg L-1; the use of 400 µL sample volumes led to a relative standard deviation (n = 5) of 3.2% and a detection limit of 0.18 mg L-1. Recovery experiments were performed with sugar cane spirits, whiskies and tequilas to evaluate the accuracy of the method, and the recovery obtained ranged from 80.5 to 112.2%.

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The porosity of volcanic rocks can be strongly affected by secondary processes such as the percolation of water and hydrothermal fluids, thus promoting crystallization of secondary minerals in the pores. With an aim to evaluate the impact of secondary mineral fillings, this paper shows the segmentation of secondary and filled pores based on the micro-CT technique. The mineralogy of the sample was analyzed with optical microscopy. Multiscale analysis was performed to determine the total porosity before and after the processes. The results show the influence on the permeability parameter caused by crystallization.

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INTRODUCTION AND OBJECTIVES: A crucial issue when appraising the performance of non-invasive markers is the limitations of the reference standard they are compared to. Digital image analysis (DIA) was suggested as a reproducible approach expressing fibrosis numerically as a proportionate area (PA) (%). We aimed to evaluate ELF test with direct reference to PA (%), thereby explore the improvement in accuracy to discriminate significant fibrosis which may actually have been underestimated by categorical pathological staging. MATERIALS AND METHODS: PA (%) data were obtained by DIA of trichrome-stained liver biopsies of 52 chronic hepatitis patients. Paired serum samples of patients and additional 36 controls were performed to measure ELF test. Diagnostic performance characteristics of ELF test was derived in predicting significant fibrosis in the patient cohort, and also, in distinguishing healthy controls from patients with significant fibrosis. RESULTS: We found an AUROC value of 0.73 for ELF to predict significant fibrosis as assessed by DIA and a lower AUROC value of 0.66 when assessed by conventional pathology. Importantly, ELF test provided considerably high diagnostic accuracy to discriminate healthy controls from patients with significant fibrosis defined by Ishak F≥2 and TPA≥5% (AUROCs 0.93 and 0.94, respectively) with optimal ELF cut-off point of 8.4 for both. CONCLUSIONS: Digital quantification could represent a better reference standard than conventional pathology allowing a better discriminatory capability for ELF test. ELF test provided high diagnostic accuracy to discriminate healthy controls from patients with significant fibrosis suggesting a role as a screening strategy in the community setting.

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In this work, we propose a method that employed a smartphone to capture images obtained from a colorimetric spot test to determine ascorbic acid (AA) in Brazilian Amazon native and exotic fruits. The spot test reaction was based on reduction of Fe(III) by AA and further complexation with 1,10-phenanthroline. After optimization, the limit of detection was 8.5 × 10-7 mol L-1. Brazilian Amazon native fruits such as bacuri, cupuaçu, muruci, yellow mombin, as well as others as cashew, mango, orange and passion fruit, were analyzed. In order to determine the accuracy of this method, iodometric titration was used; results were in close agreement with a confidence level of 95% (paired t-test). Moreover, recoveries ranged from 87.1 to 116%. The method is economic, environmentally friendly, and portable, and might useful for small producers and family agriculture businesses unable to afford specialized laboratory analysis in the north region of Brazilian Amazon.

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In this work the development and validation of analytical methodology for determination of copper in sugarcane spirit samples is carried out. The digital image based (DIB) method was applied along with spot test from the colorimetric reaction employing the RGB color model. For the determination of copper concentration, it was used the cuprizone - a bidentate organic reagent - which forms with copper a blue chelate in an alkaline medium. A linear calibration curve over the concentration range from 0.75 to 5.00mgL-1 (r2=0.9988) was obtained and limits of detection and quantification of 0.078mgL-1 and 0.26mgL-1 were acquired, respectively. For the accuracy studies, recovery percentages ranged from 98 to 104% were obtained. The comparison of cooper concentration results in sugar cane spirits using the DIB method and Flame Atomic Absorption Spectrometry as reference method showed no significant differences between both methods, which were performed using the paired t-test in 95% of confidence level. Thus, the spot test method associated with DIB allows the use of devices as digital cameras and smartphones to evaluate colorimetric reaction with low waste generation, practicality, quickness, accuracy, precision, high portability and low-cost.

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Spheres, tubes, and planar-shaped nanomaterials as Fe3O4 nanoparticles (MNPs), multiwalled carbon nanotubes (MWCNT), and graphene oxide (GO) were used for the first time to treat microfluidic paper-based analytical devices (µPADs) and create a biocompatible layer with high catalytic surface. Once glucose measurements are critical for diabetes or glycosuria detection and monitoring, the analytical performance of the proposed devices was studied by using bienzymatic colorimetric detection of this carbohydrate. The limit of detection values achieved for glucose with µPADs treated with MNPs, MWCNT, and GO were 43, 62, and 18 µM, respectively. The paper surface modification solves problems associated with the lack of homogeneity on color measurements that compromise the sensitivity and detectability levels in clinical diagnosis.

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We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.

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Hepatic fibrosis staging is based on semiquantitative scores. Digital imaging analysis (DIA) appears more accurate because fibrosis is quantified in a continuous scale. However, high cost, lack of standardization and worldwide unavailability restrict its use in clinical practice. We developed an inexpensive and widely available DIA technique for fibrosis quantification in hepatitis C, and here, we evaluate its reproducibility and correlation with semiquantitative scores, and determine the fibrosis percentage associated with septal fibrosis and cirrhosis. 282 needle biopsies staged by Ishak and METAVIR scores were included. Images of trichrome-stained sections were captured and processed using Adobe(®) Photoshop(®) CS3 and Adobe(®) Bridge(®) softwares. The percentage of fibrosis (fibrosis index) was determined by the ratio between the fibrosis area and the total sample area, expressed in pixels calculated in an automated way. An excellent correlation between DIA fibrosis index and Ishak and METAVIR scores was observed (Spearman's r = 0.95 and 0.92; P < 0.001, respectively). Excellent intra-observer reproducibility was observed in a randomly chosen subset of 39 biopsies with an intraclass correlation index of 0.99 (95% CI, 0.95-0.99). The best cut-offs associated with septal fibrosis and cirrhosis were 6% (AUROC 0.97, 95% CI, 0.95-0.99) and 27% (AUROC 1.0, 95% CI, 0.99-1), respectively. This new DIA technique had high correlation with semiquantitative scores in hepatitis C. This method is reproducible, inexpensive and available worldwide allowing its use in clinical practice. The incorporation of DIA technique provides a more complete evaluation of fibrosis adding the quantification to architectural patterns.

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A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.

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Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89 percent and 96 percent, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90 percent and obtained the maximum value for the specificity of 92 percent. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.(AU)

Cinquenta Bursa de Fabrícius (BF) foram examinadas através de microscopia óptica convencional e imagens digitais foram obtidas e processadas através do software Matlab® 6.5. Redes Neurais Artificiais (ANN) foram geradas com a utilização do software Neuroshell® Classifier, e os dados das análises óptica e digital foram comparados. A ANN classificou corretamente a maioria dos folículos, atingindo sensibilidade e especificidade de 89 por cento e 96 por cento, respectivamente. Quando os folículos foram agrupados de forma binária houve um aumento da sensibilidade para 90 por cento e obteve-se um valor máximo para a especificidade de 92 por cento. Estes resultados demonstram que o uso da análise digital de imagem associada à ANNé uma ferramenta bastante útil para a classificação patológica da depleção linfóide da BF. Além disso, fornece resultados objetivos que permitem medir a dimensão do erro classificatório, tornando possível a comparação entre distintos bancos de dados.(AU)

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Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89 percent and 96 percent, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90 percent and obtained the maximum value for the specificity of 92 percent. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

Cinquenta Bursa de Fabrícius (BF) foram examinadas através de microscopia óptica convencional e imagens digitais foram obtidas e processadas através do software Matlab® 6.5. Redes Neurais Artificiais (ANN) foram geradas com a utilização do software Neuroshell® Classifier, e os dados das análises óptica e digital foram comparados. A ANN classificou corretamente a maioria dos folículos, atingindo sensibilidade e especificidade de 89 por cento e 96 por cento, respectivamente. Quando os folículos foram agrupados de forma binária houve um aumento da sensibilidade para 90 por cento e obteve-se um valor máximo para a especificidade de 92 por cento. Estes resultados demonstram que o uso da análise digital de imagem associada à ANNé uma ferramenta bastante útil para a classificação patológica da depleção linfóide da BF. Além disso, fornece resultados objetivos que permitem medir a dimensão do erro classificatório, tornando possível a comparação entre distintos bancos de dados.

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Esse trabalho teve como objetivo avaliar a qualidade de padronização dos cones acessórios Fine Medium (FM), Medium (M) e Medium Large (ML) de cinco diferentes marcas comerciais de cones de guta-percha e de uma marca de cone Resilon, através do desenvolvimento de um método automático de processamento e análise digital de imagens. Para tal, 21 cones de cada conicidade e marca comercial foram digitalizados em um scanner com 1200 DPI, o que implica em uma precisão de 0.021 mm, e uma rotina de processamento digital das imagens foi desenvolvida para obter medições automáticas dos seguintes parâmetros: diâmetro da ponta (D0), diâmetro a 3 mm da ponta (D3), diâmetro a 16 mm da ponta (D16), comprimento (L) e conicidade (C). As medidas encontradas foram submetidas a análises estatísticas e comparações entre os grupos das médias de cada parâmetro avaliado. As especificações ANSI/ADA números 57 e 78 foram empregadas como referências para a análise da estandardização dos cones testados. De posse dos resultados, foi possível concluir que existe uma ineficaz padronização dos cones obturadores, os quais apresentaram grandes variações entre as diversas marcas testadas e dentro de uma mesma marca. O método desenvolvido para medição dos parâmetros de controle dos cones se mostrou bastante eficaz, gerando resultados confiáveis.

The present study aimed to evaluate the standardization quality of the accessory gutta-percha cones Fine Medium (FM), Medium (M) and Medium Large (ML) of five different commercial brands of gutta-percha cones and a brand of Resilon cone, through the development of an automated method of digital image processing and analysis sequence. Hence, 21 cones of each conicity and commercial brand were digitalized with a scanner with 1200 DPI, which implicates on a precision of 0,021mm and a sequence of digital image processing were developed to obtain automatic measures of the following parameters: D0, D3, D16, length (L) and conicity (C). The measures found were submitted to statistical analysis and comparisons among the groups of the means of each parameter evaluated. Numbers 57 and 78 ANSI/ADA specifications were used as references to the standardization analysis of the tested cones. Based on the results obtained it was possible to conclude that there is an inefficient standardization of obturating cones, which presented large variations among the different tested brands and in a single brand also. The method developed to measure the parameters of the obturating cones control group was very efficient, producing reliable results.

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A padronização para a fabricação de instrumentos endodônticos em aço inoxidável contribuiu para o desenvolvimento de novos aspectos geométricos. Surgiram propostas de alterações no desenho da haste helicoidal, da seção reta transversal, da ponta, da conicidade e do diâmetro na extremidade (D0). Concomitantemente, o emprego de ligas em Níquel-Titânio possibilitou a produção de instrumentos acionados a motor, largamente empregados hoje. A cada ano a indústria lança instrumentos com diversas modificações, sem, contudo, disponibilizar informações suficientes quanto às implicações clínicas destas modificações. Existe um crescente interesse no estudo dos diferentes aspectos geométricos e sua precisa metrologia. Tradicionalmente, a aferição de aspectos geométricos de instrumentos endodônticos é realizada visualmente através de microscopia ótica. Entretanto, esse procedimento visual é lento e subjetivo. Este trabalho propõe um novo método para a metrologia de instrumentos endodônticos baseado no microscópio eletrônico de varredura e na análise digital das imagens. A profundidade de campo do MEV permite obter a imagem de todo o relevo do instrumento endodôntico a uma distância de trabalho constante. Além disso, as imagens obtidas pelo detector de elétrons retro-espalhados possuem menos artefatos e sombras, tornando a obtenção e análise das imagens mais fáceis. Adicionalmente a análise das imagens permite formas de mensuração mais eficientes, com maior velocidade e qualidade. Um porta-amostras específico foi adaptado para obtenção das imagens dos instrumentos endodônticos. Ele é composto de um conector elétrico múltiplo com terminais parafusados de 12 pólos com 4 mm de diâmetro, numa base de alumínio coberta por discos de ouro. Os nichos do conector (terminais fêmeas) têm diâmetro apropriado (2,5 mm) para o encaixe dos instrumentos endodônticos. Outrossim, o posicionamento ordenado dos referidos instrumentos no conector elétrico permite a aquisição automatizada ...

The standardization of stainless steel endodontic instruments has contributed to the development of new geometric features. Many design changes in tip shape, cross section, tapering and diameters have come out. At the same time, the use of Ni-Ti alloys enabled the production of rotary instruments, which are widely employed nowadays. In fact, the industry continuously releases novel instruments with different designs. Nevertheless, not enough information is provided about these modifications and their clinical impacts. Thus, there is a growing interest in the study of these different geometric features and their accurate metrology. Traditionally, the measurement of geometric aspects in endodontic instruments is made visually under an optical microscope. However, this visual procedure is subjective and slow. This work proposes a new method for the metrology of endodontic instruments based on scanning electron microscope (SEM) and digital image analysis. The superior depth of field of the SEM allows the whole relief of an endodontic instrument to be imaged at a constant working distance. Moreover, the images from the back-scattered electrons (BSE) detector have less artifacts and shadows, making the image analysis task easier. In addition, image analysis provides efficient ways of measuring with greater speed and statistical quality. A specific sample holder was adapted for the task of imaging endodontic files. It is composed of a multiple electrical connector, a 12 pole screw terminal strip (4 mm), on an aluminum base covered by gold plates. The holes (female terminals) in the electrical connector have an appropriate diameter (2.5 mm) to fix the endodontic instruments. Besides, the ordered placement of endodontic instruments on the electrical connector allows automating image acquisition in the SEM. The gold plates produce, in BSE images, greater atomic number contrast between the gold background and the instruments. In the built sample ...(AU)

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