How to collect high quality segmentations: use human or computer drawn object boundaries?

High quality segmentations must be captured consistently for applications such as biomedical image analysis. While human drawn segmentations are often collected because they provide a consistent level of quality, computer drawn segmentations can be collected efficiently and inexpensively. In this paper, we examine how to leverage available human and computer resources to consistently create high quality segmentations. We propose a quality control methodology. We demonstrate how to apply this approach using crowdsourced and domain expert votes for the "best" segmentation from a collection of human and computer drawn segmentations for 70 objects from a public dataset and 274 objects from biomedical images. We publicly share the library of biomedical images which includes 1,879 manual annotations of the boundaries of 274 objects. We found for the 344 objects that no single segmentation source was preferred and that human annotations are not always preferred over computer annotations. These results motivated us to examine the traditional approach to evaluate segmentation algorithms, which involves comparing the segmentations produced by the algorithms to manual annotations on benchmark datasets. We found that algorithm benchmarking results change when the comparison is made to consensus-voted segmentations. Our results led us to suggest a new segmentation approach that uses machine learning to predict the optimal segmentation source and a modified segmentation evaluation approach.National Science Foundation (IIS-0910908

Biophysical interpretation of quantitative phase image

Práce se zabývá interpretací kvantitativního fázového zobrazení pomocí techniky koherencí řízené holografické mikroskopie. Vzhledem k tomu, že tato technika generuje velké množství kvantitativních fázových obrazů o nezanedbatelné velikosti, manuální analýza by byla časově náročná a neefektivní Za účelem urychlení analýzy obrazů získaných pomocí koherencí řízené holografické mikroskopie je v této práci navržena metodika automatizované interpretace kvantitativních fázových obrazů pomocí strojového učení s učitelem. Kvantitativní fázové obrazy umožňují extrakci parametrů charakterizujících distribuci suché hmoty v buňce a poskytují tak cennou informaci o buněčném chování. Cílem této práce je navrhnout metodologii pro automatizovanou klasifikaci buněk při využití této kvantitativní informace jak ze statických, tak z časosběrných kvantitativních fázových obrazů. Navržená metodika byla testována v experimentech s živými buňkami, jimiž byla vyhodnocena výkonnost klasifikace a významnost parametrů získaných z kvantitativních fázových obrazů.This work deals with the interpretation of the quantitative phase images gained by coherence-controlled holographic microscopy. Since the datasets of quantitative phase images are of substantial size, the manual analysis would be time-consuming and inefficient. In order to speed up the analysis of images gained by coherence-controlled holographic microscopy, the methodology for automated interpretation of quantitative phase images by means of supervised machine learning is proposed in this work. The quantitative phase images enable extraction of valuable features characterizing the distribution of dry mass within the cell and hence provide important information about the live cell behaviour. The aim of this work is to propose a methodology for automated classification of cells while employing the quantitative information from both the single-time-point and time-lapse quantitative phase images. The proposed methodology was tested in the experiments with live cells, where the performance of the classification was evaluated and the relevance of the features derived from the quantitative phase image was assessed.

Combining crowd worker, algorithm, and expert efforts to find boundaries of objects in images

While traditional approaches to image analysis have typically relied upon either manual annotation by experts or purely-algorithmic approaches, the rise of crowdsourcing now provides a new source of human labor to create training data or perform computations at run-time. Given this richer design space, how should we utilize algorithms, crowds, and experts to better annotate images? To answer this question for the important task of finding the boundaries of objects or regions in images, I focus on image segmentation, an important precursor to solving a variety of fundamental image analysis problems, including recognition, classification, tracking, registration, retrieval, and 3D visualization. The first part of the work includes a detailed analysis of the relative strengths and weaknesses of three different approaches to demarcate object boundaries in images: by experts, by crowdsourced laymen, and by automated computer vision algorithms. The second part of the work describes three hybrid system designs that integrate computer vision algorithms and crowdsourced laymen to demarcate boundaries in images. Experiments revealed that hybrid system designs yielded more accurate results than relying on algorithms or crowd workers alone and could yield segmentations that are indistinguishable from those created by biomedical experts. To encourage community-wide effort to continue working on developing methods and systems for image-based studies which can have real and measurable impact that benefit society at large, datasets and code are publicly-shared (http://www.cs.bu.edu/~betke/BiomedicalImageSegmentation/)

Combining crowd worker, algorithm, and expert efforts to find boundaries of objects in images

While traditional approaches to image analysis have typically relied upon either manual annotation by experts or purely-algorithmic approaches, the rise of crowdsourcing now provides a new source of human labor to create training data or perform computations at run-time. Given this richer design space, how should we utilize algorithms, crowds, and experts to better annotate images? To answer this question for the important task of finding the boundaries of objects or regions in images, I focus on image segmentation, an important precursor to solving a variety of fundamental image analysis problems, including recognition, classification, tracking, registration, retrieval, and 3D visualization. The first part of the work includes a detailed analysis of the relative strengths and weaknesses of three different approaches to demarcate object boundaries in images: by experts, by crowdsourced laymen, and by automated computer vision algorithms. The second part of the work describes three hybrid system designs that integrate computer vision algorithms and crowdsourced laymen to demarcate boundaries in images. Experiments revealed that hybrid system designs yielded more accurate results than relying on algorithms or crowd workers alone and could yield segmentations that are indistinguishable from those created by biomedical experts. To encourage community-wide effort to continue working on developing methods and systems for image-based studies which can have real and measurable impact that benefit society at large, datasets and code are publicly-shared (http://www.cs.bu.edu/~betke/BiomedicalImageSegmentation/)

SPARC, A Matricellular Protein, In A Cell Culture Model Of Heavy Metal Cadmium Induced Bladder Urothelial Carcinoma

Bladder cancer, the most common urinary tract malignancy, has environmental toxicant exposure as one of its biggest risk factors. This lab examines potential biomarkers employing an in vitro model of urothelial carcinoma using a normal bladder cell line and its malignantly transformed cells transformed counterpart, transformed by exposure to long term, low doses of cadmium (Cd2+). Initial microarray analysis determined that Secreted Protein Acidic and Rich in Cysteine (SPARC) was the most repressed gene across all transformed cell lines compared to its normal counterpart (Garrett et al., 2014, Larson et al., 2010). SPARC, an extracellular matrix glycoprotein, functions by regulating cell-matrix interactions. It has both oncogene and tumor suppressor actions depending upon the cancer with its role in bladder carcinoma remaining unclear. Previous lab results identified three key findings regarding the role of SPARC: 1) SPARC was significantly repressed following short term, low dose cadmium exposure; 2) SPARC is expressed at moderate levels in the normal transitional epithelium, however, in urothelial carcinomas, SPARC expression is drastically repressed; and 3) when SPARC was transfected into Cd2+ transformed cells with expression being ‘forced’ via a CMV promoter, heterotransplant tumors again had very little, if any, SPARC expression (Larson et al., 2010; Slusser et al., 2016). The current study is based on 3 overarching hypotheses: 1) SPARC plays a critical role in urothelial cell proliferation, migration, attachment, and spreading; 2) Cadmium transformation significantly decreases SPARC expression by silencing the promoter early in the malignant transformation process; and 3) that in urothelial tumors generated from Cd2+-transformed cell lines, SPARC is prohibitive to tumor initiation. The results of this study advance the understanding of SPARC in transformed cells by showing that SPARC promotes cell spreading which may be inhibitory to tumor initiation, necessitating its repression; preferential transcription factor binding of SOX5 compared to Sp1/Sp3 contributing to SPARC repression; and that in serial heterotransplant tumors, repression of human tumor SPARC continues along with an increase in mouse stromal SPARC. Overall, the conclusion from this research is that SPARC acts as a tumor suppressor in the UROtsa model system requiring repression for malignant transformation and tumor initiation

New algorithms for the analysis of live-cell images acquired in phase contrast microscopy

La détection et la caractérisation automatisée des cellules constituent un enjeu important dans de nombreux domaines de recherche tels que la cicatrisation, le développement de l'embryon et des cellules souches, l’immunologie, l’oncologie, l'ingénierie tissulaire et la découverte de nouveaux médicaments. Étudier le comportement cellulaire in vitro par imagerie des cellules vivantes et par le criblage à haut débit implique des milliers d'images et de vastes quantités de données. Des outils d'analyse automatisés reposant sur la vision numérique et les méthodes non-intrusives telles que la microscopie à contraste de phase (PCM) sont nécessaires. Comme les images PCM sont difficiles à analyser en raison du halo lumineux entourant les cellules et de la difficulté à distinguer les cellules individuelles, le but de ce projet était de développer des algorithmes de traitement d'image PCM dans Matlab® afin d’en tirer de l’information reliée à la morphologie cellulaire de manière automatisée. Pour développer ces algorithmes, des séries d’images de myoblastes acquises en PCM ont été générées, en faisant croître les cellules dans un milieu avec sérum bovin (SSM) ou dans un milieu sans sérum (SFM) sur plusieurs passages. La surface recouverte par les cellules a été estimée en utilisant un filtre de plage de valeurs, un seuil et une taille minimale de coupe afin d'examiner la cinétique de croissance cellulaire. Les résultats ont montré que les cellules avaient des taux de croissance similaires pour les deux milieux de culture, mais que celui-ci diminue de façon linéaire avec le nombre de passages. La méthode de transformée par ondelette continue combinée à l’analyse d'image multivariée (UWT-MIA) a été élaborée afin d’estimer la distribution de caractéristiques morphologiques des cellules (axe majeur, axe mineur, orientation et rondeur). Une analyse multivariée réalisée sur l’ensemble de la base de données (environ 1 million d’images PCM) a montré d'une manière quantitative que les myoblastes cultivés dans le milieu SFM étaient plus allongés et plus petits que ceux cultivés dans le milieu SSM. Les algorithmes développés grâce à ce projet pourraient être utilisés sur d'autres phénotypes cellulaires pour des applications de criblage à haut débit et de contrôle de cultures cellulaires.Automated cell detection and characterization is important in many research fields such as wound healing, embryo development, immune system studies, cancer research, parasite spreading, tissue engineering, stem cell research and drug research and testing. Studying in vitro cellular behavior via live-cell imaging and high-throughput screening involves thousands of images and vast amounts of data, and automated analysis tools relying on machine vision methods and non-intrusive methods such as phase contrast microscopy (PCM) are a necessity. However, there are still some challenges to overcome, since PCM images are difficult to analyze because of the bright halo surrounding the cells and blurry cell-cell boundaries when they are touching. The goal of this project was to develop image processing algorithms to analyze PCM images in an automated fashion, capable of processing large datasets of images to extract information related to cellular viability and morphology. To develop these algorithms, a large dataset of myoblasts images acquired in live-cell imaging (in PCM) was created, growing the cells in either a serum-supplemented (SSM) or a serum-free (SFM) medium over several passages. As a result, algorithms capable of computing the cell-covered surface and cellular morphological features were programmed in Matlab®. The cell-covered surface was estimated using a range filter, a threshold and a minimum cut size in order to look at the cellular growth kinetics. Results showed that the cells were growing at similar paces for both media, but their growth rate was decreasing linearly with passage number. The undecimated wavelet transform multivariate image analysis (UWT-MIA) method was developed, and was used to estimate cellular morphological features distributions (major axis, minor axis, orientation and roundness distributions) on a very large PCM image dataset using the Gabor continuous wavelet transform. Multivariate data analysis performed on the whole database (around 1 million PCM images) showed in a quantitative manner that myoblasts grown in SFM were more elongated and smaller than cells grown in SSM. The algorithms developed through this project could be used in the future on other cellular phenotypes for high-throughput screening and cell culture control applications

Classification Algorithms based on Generalized Polynomial Chaos

Classification is one of the most important tasks in process system engineering. Since most of the classification algorithms are generally based on mathematical models, they inseparably involve the quantification and propagation of model uncertainty onto the variables used for classification. Such uncertainty may originate from either a lack of knowledge of the underlying process or from the intrinsic time varying phenomena such as unmeasured disturbances and noise. Often, model uncertainty has been modeled in a probabilistic way and Monte Carlo (MC) type sampling methods have been the method of choice for quantifying the effects of uncertainty. However, MC methods may be computationally prohibitive especially for nonlinear complex systems and systems involving many variables. Alternatively, stochastic spectral methods such as the generalized polynomial chaos (gPC) expansion have emerged as a promising technique that can be used for uncertainty quantification and propagation. Such methods can approximate the stochastic variables by a truncated gPC series where the coefficients of these series can be calculated by Galerkin projection with the mathematical models describing the process. Following these steps, the gPC expansion based methods can converge much faster to a solution than MC type sampling based methods. Using the gPC based uncertainty quantification and propagation method, this current project focuses on the following three problems: (i) fault detection and diagnosis (FDD) in the presence of stochastic faults entering the system; (ii) simultaneous optimal tuning of a FDD algorithm and a feedback controller to enhance the detectability of faults while mitigating the closed loop process variability; (iii) classification of apoptotic cells versus normal cells using morphological features identified from a stochastic image segmentation algorithm in combination with machine learning techniques. The algorithms developed in this work are shown to be highly efficient in terms of computational time, improved fault diagnosis and accurate classification of apoptotic versus normal cells

Development of an image processing method for automated, non-invasive and scale-independent monitoring of adherent cell cultures

Adherent cell culture is a key experimental method for biological investigations in diverse areas such as developmental biology, drug discovery and biotechnology. Light microscopy-based methods, for example phase contrast microscopy (PCM), are routinely used for visual inspection of adherent cells cultured in transparent polymeric vessels. However, the outcome of such inspections is qualitative and highly subjective. Analytical methods that produce quantitative results can be used but often at the expense of culture integrity or viability. In this work, an imaging-based strategy to adherent cell cultures monitoring was investigated. Automated image processing and analysis of PCM images enabled quantitative measurements of key cell culture characteristics. Two types of segmentation algorithms for the detection of cellular objects on PCM images were evaluated. The first one, based on contrast filters and dynamic programming was quick (<1s per 1280×960 image) and performed well for different cell lines, over a wide range of imaging conditions. The second approach, termed ‘trainable segmentation’, was based on machine learning using a variety of image features such as local structures and symmetries. It accommodated complex segmentation tasks while maintaining low processing times (<5s per 1280×960 image). Based on the output from these segmentation algorithms, imaging-based monitoring of a large palette of cell responses was demonstrated, including proliferation, growth arrest, differentiation, and cell death. This approach is non-invasive and applicable to any transparent culture vessel, including microfabricated culture devices where a lack of suitable analytical methods often limits their applicability. This work was a significant contribution towards the establishment of robust, standardised, and affordable monitoring methods for adherent cell cultures. Finally, automated image processing was combined with computer-controlled cultures in small-scale devices. This provided a first demonstration of how adaptive culture protocols could be established; i.e. culture protocols which are based on cellular response instead of arbitrary time points