SUMMARY: Advances in 3D live cell microscopy are enabling high-resolution capture of previously unobserved processes. Unleashing the power of modern machine learning methods to fully benefit from these technologies is, however, frustrated by the difficulty of manually annotating 3D training data. MiCellAnnGELo virtual reality software offers an immersive environment for viewing and interacting with 4D microscopy data, including efficient tools for annotation. We present tools for labelling cell surfaces with a wide range of applications, including cell motility, endocytosis and transmembrane signalling. AVAILABILITY AND IMPLEMENTATION: MiCellAnnGELo employs the cross-platform (Mac/Unix/Windows) Unity game engine and is available under the MIT licence at https://github.com/CellDynamics/MiCellAnnGELo.git, together with sample data. MiCellAnnGELo can be run in desktop mode on a 2D screen or in 3D using a standard VR headset with a compatible GPU. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-resolution 3D microscopy is a fast advancing field and requires new techniques in image analysis to handle these new datasets. In this work, we focus on detailed 3D segmentation of Dictyostelium cells undergoing macropinocytosis captured on an iSPIM microscope. We propose a novel random walker-based method with a curvature-based enhancement term, with the aim of capturing fine protrusions, such as filopodia and deep invaginations, such as macropinocytotic cups, on the cell surface. We tested our method on both real and synthetic 3D image volumes, demonstrating that the inclusion of the curvature enhancement term can improve the segmentation of the aforementioned features. We show that our method performs better than other state of the art segmentation methods in 3D images of Dictyostelium cells, and performs competitively against CNN-based methods in two Cell Tracking Challenge datasets, demonstrating the ability to obtain accurate segmentations without the requirement of large training datasets. We also present an automated seeding method for microscopy data, which, combined with the curvature-enhanced random walker method, enables the segmentation of large time series with minimal input from the experimenter.

Summary: Transmembrane signalling plays important physiological roles, with G protein-coupled cell surface receptors being particularly important therapeutic targets. Fluorescent proteins are widely used to study signalling, but analyses of image time series can be challenging, in particular when cells change shape. QuimP software semi-automatically tracks spatio-temporal patterns of fluorescence at the cell membrane at high spatial resolution. This makes it a unique tool for studying transmembrane signalling, particularly during cell migration in immune or cancer cells for example. Availability and implementation: QuimP (http://warwick.ac.uk/quimp) is a set of Java plugins for Fiji/ImageJ (http://fiji.sc) installable through the Fiji Updater (http://warwick.ac.uk/quimp/wiki-pages/installation). It is compatible with Mac, Windows and Unix operating systems, requiring version >1.45 of ImageJ and Java 8. QuimP is released as open source (https://github.com/CellDynamics/QuimP) under an academic licence. Supplementary information: Supplementary data are available at Bioinformatics online.

BACKGROUND: Fundamental cellular processes such as cell movement, division or food uptake critically depend on cells being able to change shape. Fast acquisition of three-dimensional image time series has now become possible, but we lack efficient tools for analysing shape deformations in order to understand the real three-dimensional nature of shape changes. RESULTS: We present a framework for 3D+time cell shape analysis. The main contribution is three-fold: First, we develop a fast, automatic random walker method for cell segmentation. Second, a novel topology fixing method is proposed to fix segmented binary volumes without spherical topology. Third, we show that algorithms used for each individual step of the analysis pipeline (cell segmentation, topology fixing, spherical parameterization, and shape representation) are closely related to the Laplacian operator. The framework is applied to the shape analysis of neutrophil cells. CONCLUSIONS: The method we propose for cell segmentation is faster than the traditional random walker method or the level set method, and performs better on 3D time-series of neutrophil cells, which are comparatively noisy as stacks have to be acquired fast enough to account for cell motion. Our method for topology fixing outperforms the tools provided by SPHARM-MAT and SPHARM-PDM in terms of their successful fixing rates. The different tasks in the presented pipeline for 3D+time shape analysis of cells can be solved using Laplacian approaches, opening the possibility of eventually combining individual steps in order to speed up computations.