title: Normalized polarization ratios for the analysis of cell polarity.
abstract: The quantification and analysis of molecular localization in living cells is increasingly important for elucidating biological pathways, and new methods are rapidly emerging. The quantification of cell polarity has generated much interest recently, and ratiometric analysis of fluorescence microscopy images provides one means to quantify cell polarity. However, detection of fluorescence, and the ratiometric measurement, is likely to be sensitive to acquisition settings and image processing parameters. Using imaging of EGFP-expressing cells and computer simulations of variations in fluorescence ratios, we characterized the dependence of ratiometric measurements on processing parameters. This analysis showed that image settings alter polarization measurements; and that clustered localization is more susceptible to artifacts than homogeneous localization. To correct for such inconsistencies, we developed and validated a method for choosing the most appropriate analysis settings, and for incorporating internal controls to ensure fidelity of polarity measurements. This approach is applicable to testing polarity in all cells where the axis of polarity is known.
authors: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors: Raz
authors: R
authors: Shimoni
authors: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors: Kim
authors: K
authors: Pham
authors: Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors: Mohammed
authors: M
authors: Yassin
authors: Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors: Mandy J
authors: MJ
authors: Ludford-Menting
authors: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia.
authors: Min
authors: M
authors: Gu
authors: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors: Sarah M
authors: SM
authors: Russell
mesh_terms: D000465:Algorithms; D016764:Cell Polarity; D002478:Cells, Cultured; D003198:Computer Simulation; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins