Apotome 2 structured illumination slider

Fixed NIH3T3 fibroblasts were imaged under epifluorescence illumination using an upright Axio Imager.M2 microscope equipped with a 63× 1.4NA objective and with an Apotome 2 structured illumination slider (all from Zeiss). Images were acquired with a black and white CMOS camera (Hamamatsu ORCA Flash 4.0 V2) and ZEN 2 software (Zeiss). For quantification of co‐localization of the EGFP‐tagged proteins with 594‐Phalloidin in fibroblasts, we analyzed the pixel intensities along a virtual line across the cell, excluding the nucleus (ImageJ, http://imagej.nih.gov/ij). The placing of the line was done using the “actin channel”, and the experimenter was blinded to the transfections. We plotted the pixel intensities of both channels against each other and calculated the Pearson’s correlation coefficient (GraphPad Prism 7). Each individual experiment consists of 1–4 transfected wells. The person who analyzed the co‐localization and calculated Pearson’s coefficient values was blind to the transfection.
The dendritic spines of cultured hippocampal neurons were imaged using a Zeiss LSM 710 upright confocal microscope (63× 1.3NA objective) or an Axio Imager.M2 microscope (63× 1.4NA objective, Apotome 2 structured illumination slider). Image files were processed with ZEN 2012 (Carl Zeiss Microscopy GmbH), ImageJ 1.46r, and Photoshop CS4 (Adobe).