Super-Resolution Imaging Techniques for Cellular Visualization

TIRF-SIM imaging was performed on either a custom TIRF-SIM microscope at the Janelia Research Campus as previously described^{17 (link)} or a DeltaVision OMX SR (GE) microscope equipped with a 60× 1.42 NA objective. All 2D- and 3D-SIM imaging was performed on a DeltaVision OMX 3D-SIM Imaging System V4 (GE) equipped with an Olympus 60×/1.42 NA objective. Raw images were reconstructed using Softworx (Applied Precision). For 2D-SIM, a Weiner constant of 0.2 was used for every frame of processing. For 3D-SIM, a Weiner constant of 0.001 was used. Processed SIM images were zero-clipped to remove negative pixel values. Of note, some SIM images contain faint horizontal “stripes” that arise when imaging near the lower end of the CMOS camera’s dynamic range. Airyscan imaging was performed in Superresolution mode on a Zeiss LSM 880 Airyscan microscope equipped with a 63× 1.4 NA objective. Raw data was processed using Airyscan processing in “auto strength” mode (mean strength +/− S.D. = 5.5+/− 1.3) with Zen Black software version 2.3. Fig. 5A was collected on a Nikon A1R microscope equipped with a 40× 1.3 NA objective. Linear adjustments for contrast and brightness were made to images using ImageJ.