Subdiffraction-limit imaging of BRCA1 and 53BP1 in IRIF

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Subdiffraction-limit imaging of BRCA1 and 53BP1 in IRIF Subdiffraction-limit imaging of BRCA1 and 53BP1 in IRIF. (A) RPE1 cells recovered for 30 minutes following irradiation (2 Gy), were subjected to detergent pre-extraction and fixation before immunostaining for BRCA1 and 53BP1. Subdiffraction-limit imaging of BRCA1 and 53BP1 in IRIF. (A) RPE1 cells recovered for 30 minutes following irradiation (2 Gy), were subjected to detergent pre-extraction and fixation before immunostaining for BRCA1 and 53BP1. Panels represent projection images constructed from multiple CLSM Z-series images. (B) Projection images constructed from 3D-SIM acquired Z-series of cells prepared exactly as in A. (C,D) Projection (left) and 3D-rendered images (other panels) were constructed from Z-series images of the indicated cell volumes (checked box) in A and B, respectively. (E) Pixel-by-pixel analysis of colocalisation of indicated fluorescently labelled proteins was performed across whole-cell 3D-SIM Z-series by intensity correlation analysis (ICA). Representative Z-slices from each dataset are presented, and the product of the differences from the mean intensity of each pixel (PDM) is shown: positive PDM pixels (an indication of co-variance) are orange, and negative PDM pixels (an indication of inverse correlation) are blue. (F) Statistical analysis of colocalisation. ICA analysis was performed across multiple Z-series (8–32 Z-slices/cell) and standard measures of image colocalisation for each labelled antibody pair are plotted. Each dot represents a single cell. Mean and s.d. are shown. J. Ross Chapman et al. J Cell Sci 2012;125:3529-3534 © 2012. Published by The Company of Biologists Ltd