3D-SIM Imaging Data Acquisition and Reconstruction

Acquire 3D-SIM dataSingle molecule according to manufacturer’s guidelines and good imaging practices, balancing signal–noise and bleaching while correcting for spherical aberration [22 (link), 23 (link)]. Check raw data with the open source ImageJ plugin SIMcheck (Fig. 2).Fig. 2

Representative output from SIMcheck. For detailed explanation of these plots and statistics see [18 (link)]

Perform image reconstruction using commercial software that accompanies the instrument (in our case, SoftWORX by GE for the OMX V3) or an open-source alternative, such as fairSIM [24 (link)]. For multichannel imaging you will need to register channels using Tetraspec beads data and an appropriate Image registration software.

Check the quality of reconstruction using SIMcheck (Fig. 2).