Single-Molecule Localization Microscopy  •  Software Benchmarking

Real dataset • Tubulin-A647-3D

Real dataset Courtesy of Jonas Ries (EMBL)
  • 3D astigmatic image of microtubules imaged using dSTORM with a cylindrical lens (112'683 frames)
  • Microtubules in U-2 OS cells, labeled with anti-alpha tubulin primary and Alexa Fluor 647-coupled secondary antibodies.
  • Calibration data for the tubulin 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 S6)


Download may not be decompressable using the standard Windows unzip utility. In this case, use 7zip.

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

Type Modality Link to Download Format Size
ASTubulin-A647-3D-as-sequence.zipDownload as list of TIF images (ZIP)14563 Mb
ASTubulin-A647-3D-BEADS-as-sequence.zipDownload as list of TIF images (ZIP)82 Mb
ASTubulin-A647-3D-BEADS-as-stacks.tifDownload as stack, split across n files210 Mb
ASTubulin-A647-3D-stacks_1.tifDownload as stack, split across n files4377 Mb
ASTubulin-A647-3D-stacks_2.tifDownload as stack, split across n files4377 Mb
ASTubulin-A647-3D-stacks_3.tifDownload as stack, split across n files4377 Mb
ASTubulin-A647-3D-stacks_4.tifDownload as stack, split across n files4377 Mb

Analysis notes

The first 4 frames in the Tubulin beads dataset are not in the correct Z-position (due to Z-piezo travelling time) and should be ignored. In any case they are also outside of the range of meaningful signal.

Z axis: In order for easy inter-software comparison, please define frame 201 of 401 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 a small amount of Z drift (<5 nm) which can optionally 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 117 nm, Y 127 nmnm
(Astigmatism gives non-unity aspect ratio)
Image offset398.6
EM Gain100
Readout noise58.8e-
DyeAlexa 647
Wavelength665 nm emission max
Microscope objective160x/1.43-NA; Leica
Numerical aperture (NA)1.43
ADU to photon conversion factor5e-/ADU
Field of view324x271pixels
Total gain, (=EM gain/conversion factor)20ADU/e-
File formatTIFF 16-bits

Parameters of the z-stacks of beads

EM GainNo EM gain
Readout noise22.6e-
Z-step per frame10nm
ADU to photon conversion factor4.55e-/ADU
Total gain, (=1/conversion factor)0.220ADU/e-
Image offset400

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