Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis

In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis

A machine learning approach for single cell interphase cell cycle staging

The cell nucleus is a tightly regulated organelle and its architectural structure is dynamically orchestrated to maintain normal cell function. Indeed, fluctuations in nuclear size and shape are known to occur during the cell cycle and alterations in nuclear morphology are also hallmarks of many diseases including cancer. Regrettably, automated reliable tools for cell cycle staging at single cell level using in situ images are still limited. It is therefore urgent to establish accurate strategies combining bioimaging with high-content image analysis for a bona fide classification. In this study we developed a supervised machine learning method for interphase cell cycle staging of individual adherent cells using in situ fluorescence images of nuclei stained with DAPI. A Support Vector Machine (SVM) classifier operated over normalized nuclear features using more than 3500 DAPI stained nuclei. Molecular ground truth labels were obtained by automatic image processing using fluorescent ubiquitination-based cell cycle indicator (Fucci) technology. An average F1-Score of 87.7% was achieved with this framework. Furthermore, the method was validated on distinct cell types reaching recall values higher than 89%. Our method is a robust approach to identify cells in G1 or S/G2 at the individual level, with implications in research and clinical applications.This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalização (POCI), Programa Opera-cional Regional do Norte (Norte 2020) and by National Funds through the Portuguese Foundation for Science and Technology (FCT), under the projects PTDC/BBB-IMG/0283/2014, PTDC/BTM-SAL/30383/2017, LARSyS-UIDB/50009/2020, LARSyS-UID/EEA/50009/2019, NORTE-01-0145-FEDER-000029 and doctoral grant SFRH/ BD/114687/2016. The authors acknowledge the American Association of Patients with Hereditary Gastric Cancer “No Stomach for Cancer” for funding Seruca’s research and the support of the i3S Scientific Platform Advanced Light Microscopy, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122)

An open-source solution for advanced imaging flow cytometry data analysis using machine learning

Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using “user-friendly” platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data set. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery

A generic classification-based method for segmentation of nuclei in 3D images of early embryos

BACKGROUND: Studying how individual cells spatially and temporally organize within the embryo is a fundamental issue in modern developmental biology to better understand the first stages of embryogenesis. In order to perform high-throughput analyses in three-dimensional microscopic images, it is essential to be able to automatically segment, classify and track cell nuclei. Many 3D/4D segmentation and tracking algorithms have been reported in the literature. Most of them are specific to particular models or acquisition systems and often require the fine tuning of parameters. RESULTS: We present a new automatic algorithm to segment and simultaneously classify cell nuclei in 3D/4D images. Segmentation relies on training samples that are interactively provided by the user and on an iterative thresholding process. This algorithm can correctly segment nuclei even when they are touching, and remains effective under temporal and spatial intensity variations. The segmentation is coupled to a classification of nuclei according to cell cycle phases, allowing biologists to quantify the effect of genetic perturbations and drug treatments. Robust 3D geometrical shape descriptors are used as training features for classification. Segmentation and classification results of three complete datasets are presented. In our working dataset of the Caenorhabditis elegans embryo, only 21 nuclei out of 3,585 were not detected, the overall F-score for segmentation reached 0.99, and more than 95% of the nuclei were classified in the correct cell cycle phase. No merging of nuclei was found. CONCLUSION: We developed a novel generic algorithm for segmentation and classification in 3D images. The method, referred to as Adaptive Generic Iterative Thresholding Algorithm (AGITA), is freely available as an ImageJ plug-in

Context based mixture model for cell phase identification in automated fluorescence microscopy

BACKGROUND: Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task. RESULTS: The data is generated from Hela H2B GFP cells imaged during a 2-day period with images acquired 15 minutes apart using an automated time-lapse fluorescence microscopy. The patterns are described with four kinds of features, including twelve general features, Haralick texture features, Zernike moment features, and wavelet features. To generate a new set of features with more discriminate power, the commonly used feature reduction techniques are used, which include Principle Component Analysis (PCA), Linear Discriminant Analysis (LDA), Maximum Margin Criterion (MMC), Stepwise Discriminate Analysis based Feature Selection (SDAFS), and Genetic Algorithm based Feature Selection (GAFS). Then, we propose a Context Based Mixture Model (CBMM) for dealing with the time-series cell sequence information and compare it to other traditional classifiers: Support Vector Machine (SVM), Neural Network (NN), and K-Nearest Neighbor (KNN). Being a standard practice in machine learning, we systematically compare the performance of a number of common feature reduction techniques and classifiers to select an optimal combination of a feature reduction technique and a classifier. A cellular database containing 100 manually labelled subsequence is built for evaluating the performance of the classifiers. The generalization error is estimated using the cross validation technique. The experimental results show that CBMM outperforms all other classifies in identifying prophase and has the best overall performance. CONCLUSION: The application of feature reduction techniques can improve the prediction accuracy significantly. CBMM can effectively utilize the contextual information and has the best overall performance when combined with any of the previously mentioned feature reduction techniques

Cell cycle phase classification in 3D in vivo microscopy of Drosophila embryogenesis

10th International Conference on Bioinformatics - 1st ISCB Asia Joint Conference 2011, InCoB 2011/ISCB-Asia 2011: Bioinformatics - Proceedings from Asia Pacific Bioinformatics Network (APBioNet)12SUPPL. 13

ACDC: Automated Cell Detection and Counting for Time-Lapse Fluorescence Microscopy.

Advances in microscopy imaging technologies have enabled the visualization of live-cell dynamic processes using time-lapse microscopy imaging. However, modern methods exhibit several limitations related to the training phases and to time constraints, hindering their application in the laboratory practice. In this work, we present a novel method, named Automated Cell Detection and Counting (ACDC), designed for activity detection of fluorescent labeled cell nuclei in time-lapse microscopy. ACDC overcomes the limitations of the literature methods, by first applying bilateral filtering on the original image to smooth the input cell images while preserving edge sharpness, and then by exploiting the watershed transform and morphological filtering. Moreover, ACDC represents a feasible solution for the laboratory practice, as it can leverage multi-core architectures in computer clusters to efficiently handle large-scale imaging datasets. Indeed, our Parent-Workers implementation of ACDC allows to obtain up to a 3.7× speed-up compared to the sequential counterpart. ACDC was tested on two distinct cell imaging datasets to assess its accuracy and effectiveness on images with different characteristics. We achieved an accurate cell-count and nuclei segmentation without relying on large-scale annotated datasets, a result confirmed by the average Dice Similarity Coefficients of 76.84 and 88.64 and the Pearson coefficients of 0.99 and 0.96, calculated against the manual cell counting, on the two tested datasets