In our paper, we present a performance evaluation of image segmentation algorithms on microscopic image data. In spite of the existence of many algorithms for image data partitioning, there is no universal and 'the best' method yet. Moreover, images of microscopic samples can be of various character and quality which can negatively influence the performance of image segmentation algorithms. Thus, the issue of selecting suitable method for a given set of image data is of big interest. We carried out a large number of experiments with a variety of segmentation methods to evaluate the behaviour of individual approaches on the testing set of microscopic images (cross-section images taken in three different modalities from the field of art restoration). The segmentation results were assessed by several indices used for measuring the output quality of image segmentation algorithms. In the end, the benefit of segmentation combination approach is studied and applicability of achieved results on another representatives of microscopic data category - biological samples - is shown.

• Keywords
• Image analysis, image segmentation, microscopic images, performance evaluation,
• MeSH
• Algorithms * MeSH
• Microscopy * methods   MeSH
• Mice MeSH
• Image Processing, Computer-Assisted methods   standards   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.

• Keywords
• Cell segmentation, Differential contrast image, Image reconstruction, Laplacian of Gaussians, Methods comparison, Microscopy, Quantitative phase imaging,
• MeSH
• Algorithms MeSH
• Cell Fractionation methods   MeSH
• Humans MeSH
• Microscopy methods   MeSH
• Image Processing, Computer-Assisted MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The analysis and segmentation of articular cartilage magnetic resonance (MR) images belongs to one of the most commonly routine tasks in diagnostics of the musculoskeletal system of the knee area. Conventional regional segmentation methods, which are based either on the histogram partitioning (e.g., Otsu method) or clustering methods (e.g., K-means), have been frequently used for the task of regional segmentation. Such methods are well known as fast and well working in the environment, where cartilage image features are reliably recognizable. The well-known fact is that the performance of these methods is prone to the image noise and artefacts. In this context, regional segmentation strategies, driven by either genetic algorithms or selected evolutionary computing strategies, have the potential to overcome these traditional methods such as Otsu thresholding or K-means in the context of their performance. These optimization strategies consecutively generate a pyramid of a possible set of histogram thresholds, of which the quality is evaluated by using the fitness function based on Kapur's entropy maximization to find the most optimal combination of thresholds for articular cartilage segmentation. On the other hand, such optimization strategies are often computationally demanding, which is a limitation of using such methods for a stack of MR images. In this study, we publish a comprehensive analysis of the optimization methods based on fuzzy soft segmentation, driven by artificial bee colony (ABC), particle swarm optimization (PSO), Darwinian particle swarm optimization (DPSO), and a genetic algorithm for an optimal thresholding selection against the routine segmentations Otsu and K-means for analysis and the features extraction of articular cartilage from MR images. This study objectively analyzes the performance of the segmentation strategies upon variable noise with dynamic intensities to report a segmentation's robustness in various image conditions for a various number of segmentation classes (4, 7, and 10), cartilage features (area, perimeter, and skeleton) extraction preciseness against the routine segmentation strategies, and lastly the computing time, which represents an important factor of segmentation performance. We use the same settings on individual optimization strategies: 100 iterations and 50 population. This study suggests that the combination of fuzzy thresholding with an ABC algorithm gives the best performance in the comparison with other methods as from the view of the segmentation influence of additive dynamic noise influence, also for cartilage features extraction. On the other hand, using genetic algorithms for cartilage segmentation in some cases does not give a good performance. In most cases, the analyzed optimization strategies significantly overcome the routine segmentation methods except for the computing time, which is normally lower for the routine algorithms. We also publish statistical tests of significance, showing differences in the performance of individual optimization strategies against Otsu and K-means method. Lastly, as a part of this study, we publish a software environment, integrating all the methods from this study.

• Keywords
• ABC, DPSO, K-means clustering, Otsu thresholding, PSO, articular cartilage, medical image segmentation, regional segmentation,
• MeSH
• Algorithms MeSH
• Artifacts MeSH
• Cartilage, Articular * diagnostic imaging   MeSH
• Magnetic Resonance Imaging methods   MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Cluster Analysis MeSH
• Publication type
• Journal Article MeSH

This paper describes an evaluation framework that allows a standardized and quantitative comparison of IVUS lumen and media segmentation algorithms. This framework has been introduced at the MICCAI 2011 Computing and Visualization for (Intra)Vascular Imaging (CVII) workshop, comparing the results of eight teams that participated. We describe the available data-base comprising of multi-center, multi-vendor and multi-frequency IVUS datasets, their acquisition, the creation of the reference standard and the evaluation measures. The approaches address segmentation of the lumen, the media, or both borders; semi- or fully-automatic operation; and 2-D vs. 3-D methodology. Three performance measures for quantitative analysis have been proposed. The results of the evaluation indicate that segmentation of the vessel lumen and media is possible with an accuracy that is comparable to manual annotation when semi-automatic methods are used, as well as encouraging results can be obtained also in case of fully-automatic segmentation. The analysis performed in this paper also highlights the challenges in IVUS segmentation that remains to be solved.

• Keywords
• Algorithm comparison, Evaluation framework, IVUS (intravascular ultrasound), Image segmentation,
• MeSH
• Databases, Factual standards   MeSH
• Internationality MeSH
• Image Interpretation, Computer-Assisted methods   standards   MeSH
• Ultrasonography, Interventional methods   standards   MeSH
• Humans MeSH
• Coronary Artery Disease diagnostic imaging   MeSH
• Reference Values MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Practice Guidelines as Topic * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Image segmentation using bi-level thresholds works well for straightforward scenarios; however, dealing with complex images that contain multiple objects or colors presents considerable computational difficulties. Multi-level thresholding is crucial for these situations, but it also introduces a challenging optimization problem. This paper presents an improved Reptile Search Algorithm (RSA) that includes a Gbest operator to enhance its performance. The proposed method determines optimal threshold values for both grayscale and color images, utilizing entropy-based objective functions derived from the Otsu and Kapur techniques. Experiments were carried out on 16 benchmark images, which included COVID-19 scans along with standard color and grayscale images. A thorough evaluation was conducted using metrics such as the fitness function, peak signal-to-noise ratio (PSNR), structural similarity index measure (SSIM), and the Friedman ranking test. The results indicate that the proposed algorithm seems to surpass existing state-of-the-art methods, demonstrating its effectiveness and robustness in multi-level thresholding tasks.

• Keywords
• Image segmentation, Medical images, Multi-level threshold, Otsu method, Kapur method, Reptile search algorithm,
• MeSH
• Algorithms * MeSH
• COVID-19 * diagnostic imaging   virology   MeSH
• Humans MeSH
• Image Processing, Computer-Assisted * methods   MeSH
• Signal-To-Noise Ratio MeSH
• SARS-CoV-2 isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Automatic detection and segmentation of biological objects in 2D and 3D image data is central for countless biomedical research questions to be answered. While many existing computational methods are used to reduce manual labeling time, there is still a huge demand for further quality improvements of automated solutions. In the natural image domain, spatial embedding-based instance segmentation methods are known to yield high-quality results, but their utility to biomedical data is largely unexplored. Here we introduce EmbedSeg, an embedding-based instance segmentation method designed to segment instances of desired objects visible in 2D or 3D biomedical image data. We apply our method to four 2D and seven 3D benchmark datasets, showing that we either match or outperform existing state-of-the-art methods. While the 2D datasets and three of the 3D datasets are well known, we have created the required training data for four new 3D datasets, which we make publicly available online. Next to performance, also usability is important for a method to be useful. Hence, EmbedSeg is fully open source (https://github.com/juglab/EmbedSeg), offering (i) tutorial notebooks to train EmbedSeg models and use them to segment object instances in new data, and (ii) a napari plugin that can also be used for training and segmentation without requiring any programming experience. We believe that this renders EmbedSeg accessible to virtually everyone who requires high-quality instance segmentations in 2D or 3D biomedical image data.

• Keywords
• Embeddings, Instance Segmentation, Microscopy,
• MeSH
• Algorithms * MeSH
• Humans MeSH
• Microscopy * methods   MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Imaging, Three-Dimensional methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Segmentation is one of the most important steps in microscopy image analysis. Unfortunately, most of the methods use fluorescence images for this task, which is not suitable for analysis that requires a knowledge of area occupied by cells and an experimental design that does not allow necessary labeling. In this protocol, we present a simple method, based on edge detection and morphological operations, that separates total area occupied by cells from the background using only brightfield channel image. The resulting segmented picture can be further used as a mask for fluorescence quantification and other analyses. The whole procedure is carried out in open source software Fiji.

• Keywords
• Fiji, ImageJ, brightfield segmentation, cells, image analysis, microscopy,
• Publication type
• Journal Article MeSH

BACKGROUND: Manual analysis of (mini-)rhizotron (MR) images is tedious. Several methods have been proposed for semantic root segmentation based on homogeneous, single-source MR datasets. Recent advances in deep learning (DL) have enabled automated feature extraction, but comparisons of segmentation accuracy, false positives and transferability are virtually lacking. Here we compare six state-of-the-art methods and propose two improved DL models for semantic root segmentation using a large MR dataset with and without augmented data. We determine the performance of the methods on a homogeneous maize dataset, and a mixed dataset of > 8 species (mixtures), 6 soil types and 4 imaging systems. The generalisation potential of the derived DL models is determined on a distinct, unseen dataset. RESULTS: The best performance was achieved by the U-Net models; the more complex the encoder the better the accuracy and generalisation of the model. The heterogeneous mixed MR dataset was a particularly challenging for the non-U-Net techniques. Data augmentation enhanced model performance. We demonstrated the improved performance of deep meta-architectures and feature extractors, and a reduction in the number of false positives. CONCLUSIONS: Although correction factors are still required to match human labelled root lengths, neural network architectures greatly reduce the time required to compute the root length. The more complex architectures illustrate how future improvements in root segmentation within MR images can be achieved, particularly reaching higher segmentation accuracies and model generalisation when analysing real-world datasets with artefacts-limiting the need for model retraining.

• Keywords
• Automatic image segmentation, Data augmentation, Deep learning, False positives, Fine roots, Image processing, Minirhizotron, Neural networks, Root segmentation, U-Net,
• Publication type
• Journal Article MeSH

Living cell segmentation from bright-field light microscopy images is challenging due to the image complexity and temporal changes in the living cells. Recently developed deep learning (DL)-based methods became popular in medical and microscopy image segmentation tasks due to their success and promising outcomes. The main objective of this paper is to develop a deep learning, U-Net-based method to segment the living cells of the HeLa line in bright-field transmitted light microscopy. To find the most suitable architecture for our datasets, a residual attention U-Net was proposed and compared with an attention and a simple U-Net architecture. The attention mechanism highlights the remarkable features and suppresses activations in the irrelevant image regions. The residual mechanism overcomes with vanishing gradient problem. The Mean-IoU score for our datasets reaches 0.9505, 0.9524, and 0.9530 for the simple, attention, and residual attention U-Net, respectively. The most accurate semantic segmentation results was achieved in the Mean-IoU and Dice metrics by applying the residual and attention mechanisms together. The watershed method applied to this best - Residual Attention - semantic segmentation result gave the segmentation with the specific information for each cell.

• Keywords
• Cell detection, Deep learning, Microscopy image segmentation, Neural network, Semantic segmentation, Tissue segmentation, Watershed segmentation,
• MeSH
• Benchmarking MeSH
• Microscopy * MeSH
• Neural Networks, Computer MeSH
• Image Processing, Computer-Assisted * methods   MeSH
• Semantics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Although the field of sleep study has greatly developed over recent years, the most common and efficient way to detect sleep issues remains a sleep examination performed in a sleep laboratory. This examination measures several vital signals by polysomnograph during a full night's sleep using multiple sensors connected to the patient's body. Nevertheless, despite being the gold standard, the sensors and the unfamiliar environment's connection inevitably impact the quality of the patient's sleep and the examination itself. Therefore, with the novel development of accurate and affordable 3D sensing devices, new approaches for non-contact sleep study have emerged. These methods utilize different techniques to extract the same breathing parameters but with contactless methods. However, to enable reliable remote extraction, these methods require accurate identification of the basic region of interest (ROI), i.e., the patient's chest area. The lack of automated ROI segmenting of 3D time series is currently holding back the development process. We propose an automatic chest area segmentation algorithm that given a time series of 3D frames containing a sleeping patient as input outputs a segmentation image with the pixels that correspond to the chest area. Beyond significantly speeding up the development process of the non-contact methods, accurate automatic segmentation can enable a more precise feature extraction. In addition, further tests of the algorithm on existing data demonstrate its ability to improve the sensitivity of a prior solution that uses manual ROI selection. The approach is on average 46.9% more sensitive with a maximal improvement of 220% when compared to manual ROI. All mentioned can pave the way for placing non-contact algorithms as leading candidates to replace existing traditional methods used today.

• Keywords
• 3D data processing, Breathing analysis, Depth sensors, Human-machine interaction, MS Kinect data acquisition, Segmentation,
• MeSH
• Algorithms * MeSH
• Respiration MeSH
• Humans MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Polysomnography MeSH
• Sleep MeSH
• Imaging, Three-Dimensional * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH