eSRRF supplementary data The eSRRF preprint is available here: https://doi.org/10.1101/2022.04.07.487490 Find the code and documentation here: https://github.com/HenriquesLab/NanoJ-eSRRF 1a: Set01a_DNA-PAINT_COS7-MT.tif DNA-PAINT of fixed COS-7 cells with indirect immunolabeling against tubulin Pixelsize: 160 nm Exposure time: 30 ms 50 000 frames 1b: Set01b_DNA-PAINT_COS7-MT_bin100.tif Dataset 1a with 100 frames binning to effectivly increase emitter density. eSRRF analysis parameters: M=5, R=0.5, S=1 all frames 2: Set02_HeLa_ffDronpa-MAP4.tif Live-cell TIRF imaging dataset of HeLa cells expressing ffDronpa-MAP4 Pixelsize: 156 nm Exposure time: 40 ms eSRRF analysis parameters: M=4 R=2 S=4 100 frames rolling analysis gap = 50 frames 3: Set03_COS7_PrSS-mEmerald-KDEL.tif Live-cell HiLO-TIRF of COS-7 cells expressing PrSS-mEmerald-KDEL marking the ER lumen. Pixelsize: 160 nm Exposure time: 5 ms eSRRF analysis parameters: M=5 R=2 S=1 100 fr rolling analysis gap = 10 frames 4a: Set4a_MFM_HeLa_TOM20.tif Live-cell MFM of HeLa cells expressing TOM20-HaloTag labeled with JF549 Pixelsize: 160 nm Exposure time: 20 ms eSRRF analysis parameters: M=4 R=2 S=1 100 fr rolling analysis gap = 25 frames 3D axial offset = 390 nm 4b: Set4b_MFM_beadcalibration.tif MFM calibration data with TetraSpeck beads immobilized on a coverslip, Pixelsize: 160 nm z-step 60 nm 5: Set5_LLS_Jurkat_ER.zip Lattice light sheet (LLS) data of the ER in Jurkat cells. The zip-folder contains single image stacks for each z-plane: 98 z-planes with 100 fr per plane at 10 ms exposure time 276 nm step size in the z-axis and 104 nm pixelsize. Each z-plane stack was process with eSRRF followed by deskewing with the LLSM plugin (https://monash-merc.github.io/llsm/docs/fiji/index.html) to recover a single 3D volume reconstruction. eSRRF analysis parameters: M=5 R=3.5 S=2 100 frames 6: Set6_TIRF_NeuronCulture_MT_Skylan-NS.tif TIRF imaging data of cultured neuron expressing Skylan-NS tagged tubulin. Pixelsize: 64.7 nm Exposure time: 100 ms eSRRF analysis parameters: M=4 R=5 S=2 200 fr 7: Set7_SDC_U2OS_Skylan-S-Actin.tif Live-cell SDC imaging data of U2OS cells transiently expressing SkylanS-ßActin over a time course of 12 hours by acquiring substacks of 50 frames at 10 min intervalls. Pixelsize: 247.6 nm Exposure time: 500ms eSRRF analysis parameters: M=5 R=2.5 S=1 50 frames

2022