Image acquisition and data analysis were performed at room temperature using a microscope (Imager.A2; Carl Zeiss), 100×, NA 1.46 oil immersion objective lens (Carl Zeiss), a monochrome charge-coupled device camera (AxioCam MRm; Carl Zeiss), and the AxioVision Rel. 4.8 software package (Carl Zeiss).
3D-SIM was performed on a ELYRA PS.1 system (Carl Zeiss) equipped with an electron-multiplying charge-coupled device (iXON DU-885; Andor Technology) with 1,004 × 1,002 pixels at room temperature. Z stacks were taken using a 100× α-Plan Apochromat oil immersion objective with an NA of 1.46. To generate structured illumination, a grid pattern is projected onto the image plane in five different positions and at five different modulation angles to obtain high frequency information within the low frequency information captured by the optical system. For the Dylight405-, Dylight488-, Cy3-channel back computation of the lower frequencies, Fourier transformation was performed using the ZEN Structured Illumination Processing tool (Carl Zeiss) to increase the resolution in the final image. Fluorochromes used were Dylight405, Dylight488, Cy3, DAPI, EGFP, and ZsGreen.
3D-SIM was performed on a ELYRA PS.1 system (Carl Zeiss) equipped with an electron-multiplying charge-coupled device (iXON DU-885; Andor Technology) with 1,004 × 1,002 pixels at room temperature. Z stacks were taken using a 100× α-Plan Apochromat oil immersion objective with an NA of 1.46. To generate structured illumination, a grid pattern is projected onto the image plane in five different positions and at five different modulation angles to obtain high frequency information within the low frequency information captured by the optical system. For the Dylight405-, Dylight488-, Cy3-channel back computation of the lower frequencies, Fourier transformation was performed using the ZEN Structured Illumination Processing tool (Carl Zeiss) to increase the resolution in the final image. Fluorochromes used were Dylight405, Dylight488, Cy3, DAPI, EGFP, and ZsGreen.