Improved region of interest selection and colocalization analysis in three-dimensional fluorescence microscopy...

Although modern fluorescence microscopy produces detailed three-dimensional (3D) datasets, colocalization analysis and region of interest (ROI) selection is most commonly performed two-dimensionally (2D) using maximum intensity projections (MIP). However, these 2D projections exclude much of the available data. Furthermore, 2D ROI selections cannot adequately select complex 3D structures which may inadvertently lead to either the exclusion of relevant or the inclusion of irrelevant data points, consequently affecting the accuracy of the colocalization analysis. Using a virtual reality (VR) enabled system, we demonstrate that 3D visualization, sample interrogation and analysis can be achieved in a highly controlled and precise manner. We calculate several key colocalization metrics using both 2D and 3D derived super-resolved structured illumination-based data sets. Using a neuronal injury model, we investigate the change in colocalization between Tau and acetylated α-tubulin at control conditions, after 6 hours and again after 24 hours. We demonstrate that performing colocalization analysis in 3D enhances its sensitivity, leading to a greater number of statistically significant differences than could be established when using 2D methods. Moreover, by carefully delimiting the 3D structures under analysis using the 3D VR system, we were able to reveal a time dependent loss in colocalization between the Tau and microtubule network as an early event in neuronal injury. This behavior could not be reliably detected using a 2D based projection. We conclude that, using 3D colocalization analysis, biologically relevant samples can be interrogated and assessed with greater precision, thereby better exploiting the potential of fluorescence-based image analysis in biomedical research.