A bioinformatics approach for the interrogation of molecular events in single cells: transforming fluorescent time-lapse microscopy images into numbers

Abstract

Exploring and exploiting the enormous potential for pharmacological modulation of the mammalian cell cycle are key goals for basic research and drug discovery. We have developed a critical advance – the high temporal resolution monitoring of cell cycle progression enabling the tracking of single cell checkpoint transitions in a noninvasive manner even within heterogeneous populations. The green fluorescent protein (GFP)-based probe has expression, location and destruction characteristics that shadow cyclin B1 dynamics in living cells [1]. The nonperturbing stealth reporter performance has been validated on high content to high throughput detection platforms comprising multi-well high-throughput screen (HTS) imaging, single cell kinetic-tracking and multiparameter flow cytometry [1,2]. Cyclin B1-GFP tracking provides sub-phase information on cell cycle progression, cell-cycle regulator dynamics in parallel with morphological landmarks and DNA content analysis. We have developed a bioinformatics software – FluroTRAK that semi automatically tracks the continuous progression of cell cycle traverse and encodes molecular readouts in bifurcating lineages. Single cell lineages which underlie the basic concept provide an elegant assay for determining the evolving and complex interplay for tumour survival at the single cell level. Our primary premise is that a bioinformatics approach dedicated to cell-based measurements provides an essential route to determining inter-event relationships revealing novel cellular and molecular event patterns.