Improved region of interest selection and colocalization analysis in three-dimensional fluorescence microscopy samples using virtual reality

PloS One
Rensu P TheartThomas R Niesler

Abstract

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...Continue Reading

References

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Methods Mentioned

BETA
Super
dissection
fluorescence microscopy

Software Mentioned

Zeiss Zen Black
SR
SIM

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