Single-Molecule Localization Microscopy  •  Software Benchmarking

Real dataset • NPC-A647-3D

Real dataset Courtesy of Jonas Ries (EMBL)
  • 3D astigmatic dSTORM image of nuclear pore complex
  • Nup107-SNAP-BG-AF647 in U-2 OS cells, imaged using dSTORM with a cylindrical lens (150'000 frames).
  • Calibration data for the dataset. Fluorescent beads adsorbed to a glass coverslide in water are stepped in Z at high SNR
  • Li et al., Nature Methods 15 2018. (raw data of Figure S9)



TIFF Stacks are split across multiple files due to TIFF 4GB file size limit.

Type Modality Link to Download Format Size
ASNPC-A647-3D-BEADS-as-sequence.zipDownload as list of TIF images (ZIP)7 Mb
ASNPC-A647-3D-BEADS-as-stacks.tifdownload as stack, split across 2 files22 Mb
ASNPC-A647-3D-stack_1.tifdownload as stack, split across 2 files3641 Mb
ASNPC-A647-3D-stack_2.tifdownload as stack, split across 2 files3642 Mb

Analysis notes

Z axis: In order for easy inter-software comparison, please define frame 21 of 41 in the beads dataset as frame Z=0nm. Frame numbers smaller than the z = 0 frame number correspond to negative Z positions. This is an arbitrarily chosen zero for the z dimension of the coordinate system, and may not correspond to what you determine as the in-focus frame.

Drift: There is XY drift which requires correction for highest resolution. There is also 25 nm of Z drift which should be corrected.

Temporal grouping: Data should be temporally grouped for highest resolution.

Submission notes

Please indicate on submission:

Parameters of the sequence

Exposure time15ms
Pixelsize, sample planeX 132 nm, Y 142 nmnm
(Astigmatism gives non-unity aspect ratio)
Image offset196
EM Gain200
Readout noise175.6e-
DyeAlexa 647
Wavelength665 nm emission max
Microscope objective60x NA1.49 Nikon objective
Numerical aperture (NA)1.49
ADU to photon conversion factor15.8e-/ADU
Total gain, (=EM gain/conversion factor)12.7ADU/e-
File formatTIFF 16-bits

Parameters of the z-stacks of beads

EM GainNo EM gain
Readout noise7.66e-
Z-step per frame50nm
ADU to photon conversion factor2.85e-/ADU
Total gain, (=1/conversion factor)0.351ADU/e-
Image offset200

© 2018 Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
Last update: 30 Nov 2018