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)

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Download

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

CameraEMCCD
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-
Frames150'000
File formatTIFF 16-bits

Parameters of the z-stacks of beads

CameraEMCCD
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