Free Image Analysis Software Research Articles

The feasibility study on the colorimetric measurement of pH under acidic conditions using pH indicator papers and image analysis

ABSTRACT This study investigates the colorimetric measurement of pH 1–3 using both RGB (red, green, and blue) and CMY (cyan, magenta, and yellow) color systems to address reliable pH measurement methods in aqueous solutions. The experiment utilized pH indicator papers to measure solutions with pH values ranging from 1 to 3. Six hundred measurements were taken for each pH level, and the resulting color changes were analyzed using a free image analysis software provided by the Korean Agency for Technology and Standards. Key findings from the RGB analysis show that for pH 1, R values predominantly fell between 172 and 227, while at pH 3, R values significantly decreased to a range of 71 to 144. In the CMY analysis, cyan values were consistently zero across all pH levels, while magenta and yellow values exhibited varying distributions depending on the pH. The study revealed that the R value in the RGB system is particularly useful for distinguishing between pH 2 and 3, while the M value in the CMY system proved effective in differentiating between pH 1 and 2. These results demonstrate the potential of colorimetric analysis using both RGB and CMY systems as a simple, cost-effective, and reliable method for pH measurement.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Detection and Quantification of Calcium Ions in the Endoplasmic Reticulum and Cytoplasm of Cultured Cells Using Fluorescent Reporter Proteins and ImageJ Software.

This protocol describes a method for detecting and quantifying calcium ions in the endoplasmic reticulum (ER) and cytoplasm of cultured cells using fluorescent reporter proteins and ImageJ software. Genetically engineered fluorescent reporter proteins, such as G-CEPIA1er and GCaMP6f, localize to intracellular regions of interest (i.e., ER and cytoplasm) and emit green fluorescence upon binding to calcium ions. In this way, the fluorescence brightness of cells transfected with expression vectors for these reporters reflects the calcium ion concentration in each intracellular region. Here, we describe procedures for observing cultured cells expressing these fluorescent reporters under a fluorescence microscope, analyzing the obtained image using the free image analysis software ImageJ (https://imagej.net/ij/index.html), and determining the average fluorescence brightness of multiple cells present in the image. The current method allows us to quickly and easily quantify calcium ions on an image containing multiple cells and to determine whether there are relative differences in intracellular calcium ion concentration among experiments with different conditions. Key features Detection and quantification of calcium ions in the ER and cytoplasm using fluorescent reporter proteins Quick and easy verification of measurement results using ImageJ Simultaneous comparison between various experimental conditions (drug treatment, mutants, etc.).

Investigation of Different Free Image Analysis Software for High-Throughput Droplet Detection.

Droplet microfluidics has revealed innovative strategies in biology and chemistry. This advancement has delivered novel quantification methods, such as droplet digital polymerase chain reaction (ddPCR) and an antibiotic heteroresistance analysis tool. For droplet analysis, researchers often use image-based detection techniques. Unfortunately, the analysis of images may require specific tools or programming skills to produce the expected results. In order to address the issue, we explore the potential use of standalone freely available software to perform image-based droplet detection. We select the four most popular software and classify them into rule-based and machine learning-based types after assessing the software’s modules. We test and evaluate the software’s (i) ability to detect droplets, (ii) accuracy and precision, and (iii) overall components and supporting material. In our experimental setting, we find that the rule-based type of software is better suited for image-based droplet detection. The rule-based type of software also has a simpler workflow or pipeline, especially aimed for non-experienced users. In our case, CellProfiler (CP) offers the most user-friendly experience for both single image and batch processing analyses.

Curiosity-Based Biophysics Projects in a High School Setting with Graduate Student Mentorship

Curiosity-Based Biophysics Projects in a High School Setting with Graduate Student Mentorship

Fibroblast Gap-closure Assay-Microscopy-based in vitro Assay Measuring the Migration of Murine Fibroblasts.

Pulmonary fibrosis is characterized by pathological scaring of the lung. Similar to other fibrotic diseases, scar formation is driven by excessive extracellular matrix deposition by activated, proliferative, and migratory fibroblasts. Currently, the two most widely used chemotaxis and cell migration assays are the scratch assay and the transmembrane invasion assay. Here we present a gap closure assay that employs commercially available cell lines, equipment and reagents and is time efficient as well as straightforward. The protocol uses an Oris pro cell migration assay 96-well plate with a dissolvable plug in the center of each well to create a cell free area at the time of seeding. Cell repopulation of the empty zone is captured via light microscopy at different time points and quantified with free image analysis software. The clear advantages of this assay in comparison to similar protocols are the use of uncomplicated cell culture methods and the ability to image the experiment throughout.

A comparison of ground-based methods for estimating canopy closure for use in phenology research

Climate change is influencing tree phenology, causing earlier and more prolonged canopy closure in temperate forests. Canopy closure is closely associated with understorey light, so shifts in its timing have wide-reaching consequences for ecological processes in the understorey. Widespread monitoring of forest canopies through time is needed to understand changes in light availability during spring in particular. Canopy openness, derived from hemispherical photography, has frequently been used as a proxy for understorey light. However, hemispherical photography is relatively resource intensive, so we tested a range of inexpensive alternatives for monitoring variability in canopy closure (visual estimation, canopy scope, smartphone photography, smartphone photography with fisheye attachment; and image analysis with specialist hemispherical photography software or with simpler, open access image analysis software). Smartphone photography with an inexpensive fisheye lens attachment proved the most reliable estimator of canopy closure. We found no significant difference in canopy estimations from three widely-owned smartphone models with differing resolutions and fields of view, and no significant effect of camera operator on the results. ImageJ, a free image analysis software, detected canopy variability in a similar way to HemiView specialist hemispherical photography software. We recommend a combination of smartphone photography with fisheye attachment and analysis with ImageJ for identifying changes in the timing of canopy closure (but not for estimating absolute canopy closure). We discuss how large-scale citizen science using this approach could generate meaningful and comparative data on the timings of canopy closure in different forests, year-to-year.

Optical Coherence Tomography Angiography to Distinguish Changes of Choroidal Neovascularization after Anti-VEGF Therapy: Monthly Loading Dose versus Pro Re Nata Regimen.

Purpose To compare the qualitative and quantitative choroidal neovascularization (CNV) changes after antivascular endothelial growth factor (anti-VEGF) therapy in treatment-naïve and treated eyes with age-related macular degeneration (AMD) using optical coherence tomography angiography (OCTA). Methods Consecutive patients with neovascular AMD underwent multimodal imaging, including OCTA (AngioPlex, CIRRUS HD-OCT model 5000; Carl Zeiss Meditec, Inc., Dublin, OH) at baseline and at three monthly follow-up visits. Treatment-naive AMD patients undergoing anti-VEGF loading phase were included in group A, while treated patients were included in group B. Qualitative and quantitative OCTA analyses were performed on outer retina to choriocapillaris (ORCC) slab. CNV size was measured using a free image analysis software (ImageJ, open-source imaging processing software, 2.0.0). Results Twenty-five eyes of 25 patients were enrolled in our study (mean age 78.32 ± 6.8 years): 13 treatment-naïve eyes in group A and 12 treated eyes in group B. While qualitative analysis revealed no significant differences from baseline to follow-up in the two groups, quantitative analysis showed in group A a significant decrease in lesion area (P = 0.023); in group B, no significant change in the lesion area was observed during anti-VEGF therapy (P = 0.93). Conclusion Treatment-naïve and treated eyes with CNV secondary to neovascular AMD respond differently to anti-VEGF therapy. This should be taken into account when using OCTA for CNV follow-up or planning therapeutic strategies.

Image analysis of interarytenoid area to detect cases of Laryngopharyngeal Reflux: An objective method

PurposeTo diagnose Laryngopharyngeal Reflux by observing colour (Red, Green, Blue) at the interarytenoid area during 70° laryngeal endoscopy. Materials and methodsEndoscopic images from 50 normal controls and 50 patients of LPR were obtained in this observational study. LPR patients were selected on the basis of RSI and RFS. Images were analysed using ImageJ, a free image analysis software, developed by the National Institute of Health (NIH). Colour changes in the form of RGB (red, green, blue) values were calculated and analysed at the interarytenoid area. The values in the normal and patient group were compared and correlated with RSI and RFS. ResultsRGB values of the LPR group and the normal group were statistically different (P value<0.01). Strong correlation was also found between R and G values and both RFS and RSI. However, no correlation was seen with B values. ConclusionImage analysis is an easy, economical and objective method to diagnose LPR.

Solid glucose biosensor integrated in a multi-well microplate coupled to a camera-based detector: Application to the multiple analysis of human serum samples

Abstract In the present work, a biosensor adapted in a 96-well microplate has been coupled with a smartphone-based photometer in order to develop a low-cost colorimetric multi-sample dispositive. The strength of this biosensing system is based on the integration of the biosensor into the 96-well microplate and the use of a smartphone and free image analysis software as a microplate reader. The performance of the proposed biosensor has been demonstrated to determine glucose in several human serum samples. This method is simple, cost-effective, sensitive and selective for the determination of glucose in serum, with detection limits of 1.8 mg/dL and a good linearity over the range 6–88 mg/dL. Precision was also satisfactory (relative standard deviation, %RSD

Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning: Detection of apoptosis in renal tissue.

The current study proposes an automated machine learning approach for the quantification of cells in cell death pathways according to DNA fragmentation. A total of 17 images of kidney histological slide samples from male Wistar rats were used. The slides were photographed using an Axio Zeiss Vert.A1 microscope with a 40x objective lens coupled with an Axio Cam MRC Zeiss camera and Zen 2012 software. The images were analyzed using CellProfiler (version 2.1.1) and CellProfiler Analyst open-source software. Out of the 10,378 objects, 4970 (47,9%) were identified as TUNEL positive, and 5408 (52,1%) were identified as TUNEL negative. On average, the sensitivity and specificity values of the machine learning approach were 0.80 and 0.77, respectively. Image cytometry provides a quantitative analytical alternative to the more traditional qualitative methods more commonly used in studies.

High-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application – SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary “Manual Initialize” and “Hand Draw” tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application – SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary “Manual Initialize” and “Hand Draw” tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

A simple and reliable methodology to detect egg white in art samples

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.

OBJECT-BASED IMAGE ANALYSIS OF WORLDVIEW-2 SATELLITE DATA FOR THE CLASSIFICATION OF MANGROVE AREAS IN THE CITY OF SÃO LUÍS, MARANHÃO STATE, BRAZIL

Abstract. Taking into account the importance of mangrove environments for the biodiversity of coastal areas, the objective of this paper is to classify the different types of irregular human occupation on the areas of mangrove vegetation in São Luis, capital of Maranhão State, Brazil, considering the OBIA (Object-based Image Analysis) approach with WorldView-2 satellite data and using InterIMAGE, a free image analysis software. A methodology for the study of the area covered by mangroves at the northern portion of the city was proposed to identify the main targets of this area, such as: marsh areas (known locally as Apicum), mangrove forests, tidal channels, blockhouses (irregular constructions), embankments, paved streets and different condominiums. Initially a databank including information on the main types of occupation and environments was established for the area under study. An image fusion (multispectral bands with panchromatic band) was done, to improve the information content of WorldView-2 data. Following an ortho-rectification was made with the dataset used, in order to compare with cartographical data from the municipality, using Ground Control Points (GCPs) collected during field survey. Using the data mining software GEODMA, a series of attributes which characterize the targets of interest was established. Afterwards the classes were structured, a knowledge model was created and the classification performed. The OBIA approach eased mapping of such sensitive areas, showing the irregular occupations and embankments of mangrove forests, reducing its area and damaging the marine biodiversity.

Evaluation of the antifungal properties of chitosan coating on cut apples using a non‐invasive image analysis technique

AbstractImage analysis techniques have often been chosen as fundamental tools for evaluating the surface and/or structure of organic and inorganic materials over a broad range of scales. In the study reported, a simple image capture system comprising a commercial desktop scanner combined with free image analysis software was used to evaluate the efficiency of antifungal activity of commercial medium‐molecular‐weight chitosan as an edible coating on sliced apples. Fungal growth rate (predominantly Penicillium sp. and Alternaria sp.) was assessed via images acquired twice daily and binary‐transformed for quantitative analysis. The inhibitory effect of chitosan coating on the mycelial fungi growth on cut apple surfaces was confirmed. Chitosan deposited on glass slides was used to simulate film formation and its structure was characterized using atomic force microscopy, revealing a thin porous formation. Copyright © 2011 Society of Chemical Industry

A practical guide to evaluating colocalization in biological microscopy.

Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

Quantitative immunohistochemical examination of the local cellular reactions following implantation of biomaterials

Examining the biocompatibility of implant materials includes the in vivo investigation of the local tissue response following implantation in experimental animals. By contrast to qualitative and semi-quantitative approaches often used in this field, a quantitative technique would facilitate a more accurate determination and better comparability of different studies. Therefore, this study aimed at evaluating the applicability of the free image analysis software ImageJ for fast, easy and reproducible quantification of the tissue response following implantation of titanium samples in rats with subsequent immunohistochemical examination of peri-implant tissue samples for monocytes and macrophages (ED1) and MHC class II positive antigen presenting cells (OX6). The quantification of positively stained cells in the vicinity of the implant pockets was based on a grid-supported manual count carried out using two ImageJ plugins (CellCounter, Grid) and resulted in a mean coefficient of variation of 13.8% (ED1) and 19.6% (OX6) between different investigators and 10.0% (ED1) and 13.8% (OX6) for repeated counting by the same investigator. In conclusion, ImageJ was found to be suitable for morphometric evaluation of the tissue response following implantation, particularly the analysis of discrete cellular events at the tissue-biomaterial interface. The procedure which was used is described in detail, and its advantages and disadvantages are discussed.

Validation of multi‐detector computed tomography as a non‐invasive method for measuring ovarian volume in macaques (Macaca fascicularis)

The purpose of this study was to validate low radiation dose, contrast-enhanced, multi-detector computed tomography (MDCT) as a non-invasive method for measuring ovarian volume in macaques. Computed tomography scans of four known-volume phantoms and nine mature female cynomolgus macaques were acquired using a previously described, low radiation dose scanning protocol, intravenous contrast enhancement, and a 32-slice MDCT scanner. Immediately following MDCT, ovaries were surgically removed and the ovarian weights were measured. The ovarian volumes were determined using water displacement. A veterinary radiologist who was unaware of actual volumes measured ovarian CT volumes three times, using a laptop computer, pen display tablet, hand-traced regions of interest, and free image analysis software. A statistician selected and performed all tests comparing the actual and CT data. Ovaries were successfully located in all MDCT scans. The iliac arteries and veins, uterus, fallopian tubes, cervix, ureters, urinary bladder, rectum, and colon were also consistently visualized. Large antral follicles were detected in six ovaries. Phantom mean CT volume was 0.702+/-SD 0.504 cc and the mean actual volume was 0.743+/-SD 0.526 cc. Ovary mean CT volume was 0.258+/-SD 0.159 cc and mean water displacement volume was 0.257+/-SD 0.145 cc. For phantoms, the mean coefficient of variation for CT volumes was 2.5%. For ovaries, the least squares mean coefficient of variation for CT volumes was 5.4%. The ovarian CT volume was significantly associated with actual ovarian volume (ICC coefficient 0.79, regression coefficient 0.5, P=0.0006) and the actual ovarian weight (ICC coefficient 0.62, regression coefficient 0.6, P=0.015). There was no association between the CT volume accuracy and mean ovarian CT density (degree of intravenous contrast enhancement), and there was no proportional or fixed bias in the CT volume measurements. Findings from this study indicate that MDCT is a valid non-invasive technique for measuring the ovarian volume in macaques.

Procedures for efficiently producing high-quality fecundity data on a small budget

Though fecundity estimation has typically been very time consuming, efficient methods have recently been developed. Building on these developments, we describe procedures for efficiently producing precise, accurate, fecundity estimates for highly fecund Atlantic cod, using a low-cost laboratory setup. Capturing images with a standard flatbed scanner and analyzing them with free image analysis software (total equipment cost ≈ $100) we were able to process individual samples in as little as 5 min. We show a strong relationship between mean oocyte diameter (range 200–900 μm) and oocyte density ( r 2 = 0.971, n = 50), and thus are able to estimate fecundity using gravimetric and auto-diametric methods. Testing the precision of the auto-diametric method we find estimates produced by both methods to be highly correlated ( r 2 = 0.961, n = 26). The success of these methods should assure fisheries scientists that fecundity estimation is no longer the daunting task it once was, and catalyze further production of fecundity data.