eSRRF - Supplementary Data

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