Temporal Evaluation of the Surface Area of Treated Skin Ulcers Caused by Cutaneous Leishmaniasis and Relation with Optical Parameters in an Animal Model: A Proof of Concept.

Cutaneous leishmaniasis (CL) is a neglected disease caused by an intracellular parasite of the Leishmania genus. CL lacks tools that allow its understanding and treatment follow-up. This article presents the use of metrical and optical tools for the analysis of the temporal evolution of treated skin ulcers caused by CL in an animal model. Leishmania braziliensis and L. panamensis were experimentally inoculated in golden hamsters, which were treated with experimental and commercial drugs. The temporal evolution was monitored by means of ulcers' surface areas, as well as absorption and scattering optical parameters. Ulcers' surface areas were obtained via photogrammetry, which is a procedure that allowed for 3D modeling of the ulcer using specialized software. Optical parameters were obtained from a spectroscopy study, representing the cutaneous tissue's biological components. A one-way ANOVA analysis was conducted to identify relationships between both the ulcers' areas and optical parameters. As a result, ulcers' surface areas were found to be related to the following optical parameters: epidermis thickness, collagen, keratinocytes, volume-fraction of blood, and oxygen saturation. This study is a proof of concept that shows that optical parameters could be associated with metrical ones, giving a more reliable concept during the assessment of a skin ulcer's healing.

An Optical Diffuse Reflectance Model for the Characterization of a Si Wafer with an Evaporated SiO2 Layer.

Thin films are a type of coating that have a very wide spectrum of applications. They may be used as single layers or composed in multilayer stacks, which significantly extend their applications. One of the most commonly used material for thin films is silicon dioxide, SiO2. Although there are other tools that can be used to measure the thickness of SiO2 films, these tools are very complex and sophisticated. In this article, we propose the use of an exponential two-layer light-material interaction model, throughout its diffuse reflectance spectra, as an alternative for the measurement of the thickness of evaporated SiO2 on Si wafers. The proposed model is evaluated experimentally by means of a 980-nm-thick SiO2 layer evaporated on a Si wafer. The results show that the proposed model has a strong correlation with the thickness measurements obtained using commercial equipment.

Estimation of Biological Parameters of Cutaneous Ulcers Caused by Leishmaniasis in an Animal Model Using Diffuse Reflectance Spectroscopy.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that requires novel tools for its understanding, diagnosis, and treatment follow-up. In the cases of other cutaneous pathologies, such as cancer or cutaneous ulcers due to diabetes, optical diffuse reflectance-based tools and methods are widely used for the investigation of those illnesses. These types of tools and methods offer the possibility to develop portable diagnosis and treatment follow-up systems. In this article, we propose the use of a three-layer diffuse reflectance model for the study of the formation of cutaneous ulcers caused by CL. The proposed model together with an inverse-modeling procedure were used in the evaluation of diffuse-reflectance spectral signatures acquired from cutaneous ulcers formed in the dorsal area of 21 golden hamsters inoculated with Leishmanisis braziliensis. As result, the quantification of the model's variables related to the main biological parameters of skin were obtained, such as: diameter and volumetric fraction of keratinocytes, collagen; volumetric fraction of hemoglobin, and oxygen saturation. Those parameters show statistically significant differences among the different stages of the CL ulcer formation. We found that these differences are coherent with histopathological manifestations reported in the literature for the main phases of CL formation.

Performance Characterization of an xy-Stage Applied to Micrometric Laser Direct Writing Lithography.

This article concerns the characterization of the stability and performance of a motorized stage used in laser direct writing lithography. The system was built from commercial components and commanded by G-code. Measurements use a pseudo-periodic-pattern (PPP) observed by a camera and image processing is based on Fourier transform and phase measurement methods. The results report that the built system has a stability against vibrations determined by peak-valley deviations of 65 nm and 26 nm in the x and y directions, respectively, with a standard deviation of 10 nm in both directions. When the xy-stage is in movement, it works with a resolution of 0.36 µm, which is an acceptable value for most of research and development (R and D) microtechnology developments in which the typical feature size used is in the micrometer range.

Diffuse Reflectance Parameters of Treated Leishmaniasis Cutaneous Ulcers and Association with Histopathologies in an Animal Model: A Proof of Concept.

Cutaneous leishmaniasis (CL) is a parasitic disease that produces chronic skin ulcers. Although it has a worldwide presence, it is a neglected disease that still requires novel tools for its management. In order to study the use of optical tools in CL, this article presents a preliminary study of the correlation between CL histopathological and optical parameters. Optical parameters correspond to absorption and scattering coefficients obtained from diffuse reflectance spectra of treated CL in golden hamsters. Independently, histopathological data were collected from the same hamsters. As a result, after Spearman correlation and the Kruskal-Wallis test, inverse correlation was found between absorption/scattering optical parameters and inflammatory histopathological values, such as the scattering parameter related to the diameter of fibroblasts with the histopathological parameters of fibrosis, polymorphonuclear neutrophils, lymphocytes, plasmocytes, hyperplasia, and Leishmania, and the absorption parameter oxygen saturation showed a relation with the granulation tissue histopathological parameter. These correlations agree with the expected behavior of tissue composition during the healing process in CL. The results correspond to a proof of concept that shows that optical diffuse reflectance-based tools and methods could be considered as an alternative to assist in CL diagnosis and treatment follow-up.

Accurate Region-of-Interest Recovery Improves the Measurement of the Cell Migration Rate in the In Vitro Wound Healing Assay.

The wound healing assay is widely used for the quantitative analysis of highly regulated cellular events. In this essay, a wound is voluntarily produced on a confluent cell monolayer, and then the rate of wound reduction (WR) is characterized by processing images of the same regions of interest (ROIs) recorded at different time intervals. In this method, sharp-image ROI recovery is indispensable to compensate for displacements of the cell cultures due either to the exploration of multiple sites of the same culture or to transfers from the microscope stage to a cell incubator. ROI recovery is usually done manually and, despite a low-magnification microscope objective is generally used (10x), repositioning imperfections constitute a major source of errors detrimental to the WR measurement accuracy. We address this ROI recovery issue by using pseudoperiodic patterns fixed onto the cell culture dishes, allowing the easy localization of ROIs and the accurate quantification of positioning errors. The method is applied to a tumor-derived cell line, and the WR rates are measured by means of two different image processing software. Sharp ROI recovery based on the proposed method is found to improve significantly the accuracy of the WR measurement and the positioning under the microscope.

Bio-EdIP: An automatic approach for in vitro cell confluence images quantification.

BACKGROUND AND OBJECTIVES: Cell imaging is a widely-employed technique to analyze multiple biological processes. Therefore, simple, accurate and quantitative tools are needed to understand cellular events. For this purpose, Bio-EdIP was developed as a user-friendly tool to quantify confluence levels using cell culture images. METHODS: The proposed algorithm combines a pre-processing step with subsequent stages that involve local processing techniques and a morphological reconstruction-based segmentation algorithm. Segmentation performance was assessed in three constructed image sets, comparing F-measure scores and AUC values (ROC analysis) for Bio-EdIP, its previous version and TScratch. Furthermore, segmentation results were compared with published algorithms using eight public benchmarks. RESULTS: Bio-EdIP automatically segmented cell-free regions from images of in vitro cell culture. Based on mean F-measure scores and ROC analysis, Bio-EdIP conserved a high performance regardless of image characteristics of the constructed dataset, when compared with its previous version and TScratch. Although acquisition quality of the public dataset affected Bio-EdIP segmentation, performance was better in two out of eight public sets. CONCLUSIONS: Bio-EdIP is a user-friendly interface, which is useful for the automatic analysis of confluence levels and cell growth processes using in vitro cell culture images. Here, we also presented new manually annotated data for algorithms evaluation.

Blind source separation of ex-vivo aorta tissue multispectral images.

Blind Source Separation methods (BSS) aim for the decomposition of a given signal in its main components or source signals. Those techniques have been widely used in the literature for the analysis of biomedical images, in order to extract the main components of an organ or tissue under study. The analysis of skin images for the extraction of melanin and hemoglobin is an example of the use of BSS. This paper presents a proof of concept of the use of source separation of ex-vivo aorta tissue multispectral Images. The images are acquired with an interference filter-based imaging system. The images are processed by means of two algorithms: Independent Components analysis and Non-negative Matrix Factorization. In both cases, it is possible to obtain maps that quantify the concentration of the main chromophores present in aortic tissue. Also, the algorithms allow for spectral absorbance of the main tissue components. Those spectral signatures were compared against the theoretical ones by using correlation coefficients. Those coefficients report values close to 0.9, which is a good estimator of the method's performance. Also, correlation coefficients lead to the identification of the concentration maps according to the evaluated chromophore. The results suggest that Multi/hyper-spectral systems together with image processing techniques is a potential tool for the analysis of cardiovascular tissue.