Project development P4

Improving throughput

The work on high-throughput screening platform development continued with further improvement of the automated fluid delivery system FCS/FCCS that utilizes microfluidic imaging chambers for high-throughput correlated and multimodal microscopy on both the Leica SP5 SMD and Zeiss LSM780 ConFoCor3 imaging system using Micropilot or Micronaut software plugins. We have also focused on further development and optimization of 384 well micro-plate based screening approach for drug sensitivity and resistance testing (DSRT) as well as siRNA screening.

Maximizing content, data processing, modeling and query

Available high-throughput image analysis software offer efficient algorithms for analysis of single time-point assays while the existing tools for the analysis of cellular dynamics in multi-dimensional large-scale imaging are very limited. During our fourth year, we developed the software Micronaut, which provides an extended and improved interface between the software controlling a confocal microscope and the computer vision / machine learning methods. The EBImage software package for R was further enriched with tools to transform images, segment cells and extract quantitative cellular descriptors. The open source standard format for high-content screening data, CellH5 has been extended to further include common analysis tasks such as dimension reduction techniques and unsupervised learning methods on extracted morphometric cell features. We continued to improve software (WP5) that performs primary statistical analysis of complex data along with quality assessment and significance analysis developing the RBioFormats software package that provides an interface between the powerful statistical and visualization environment R and the Bio-Formats microscopy image formats reader.

Development and application of modelling methods for systems microscopy

Data derived from a number of different experimental set-ups have been used to commence bold modeling projects that will ultimately serve to describe the dynamic processes of cell migration and division. During P4, we have been testing and further exploring an approach to measure the heterogeneity of single cell descriptors in a population of cells from single-cell RNA-Seq data to also make it applicable to cellular descriptors derived from microscopy imaging. We made tremendous advances in the area of mechanistic modelling on protein localization on steady-state structures with the development of a third original method to calculate the localization of molecules transported by filaments.   

Standardization measures

Development of compatibility and exchangeability of data from the packages developed by the NoE members (original multidimensional images and segmentation masks, analyzed features and statistical results) continued by including import/export modules based on HDF5 database structures and OME image file standards and the agreed common nomenclature. This allows application of complementary software (e.g. screens analysis by scores based on multidimensional statistics, by supervised or by unsupervised machine learning classification) .

Database Development

The Cellular Phenotype Database (CPD) running on a production server since 2013 it now contains 10 datasets associated with scientific publications. Phenotypes associated with these 10 studies have been mapped to Cellular Microscopy Phenotype Ontology (CMPO) terms and ontology-enabled query has been implemented through the CPD user interface for phenotype search and browsing.

The biological systems

The characteristics of cancer biology and its clinical consequences render a strong case for studying cell division and migration at the systems level. During the fourth project period we continued to work on a tertiary RNAi screens and validated the earlier identified candidate regulator of chromosome-chromosome adhesion. Further, we complemented the RNAi screening method by recently developed technologies based on CRISPR/Cas9 gene editing. Screening activities searching for genes whose knockdown affects individual or collective cell migration led to the validation of highly promising hits. Super-resolution studies using dual color characterization of adhesion sites were performed which shed new light on the molecular architecture of the adhesion sites.

Seventh Framework Programme

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