Single-Molecule Localization Microscopy  •  Software Benchmarking

Collection of reference datasets

The benchmarking of SMLM software package mainly relies on the usage of common reference datasets, in particular synthetic datasets with known ground-truth.

Conditions of use These reference datasets are designed to be largely used by the developpers to validate software and by the users to check a software. Reference: Sage et al. Quantitative evaluation of software packages for SMLM, Nature Methods 12, 2015.

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Eye

Datasets » Artificial Datasets » Eye

seashell
This datasets is an artificial structures of 4 tubes (1nm of radius) in a field of view 38.4 * 38.4 μm. Only 300 activated fluorophores in 40 frames. It is an ideal dataset: no perturbation, no additional noise.
Version: 2.0 dated 29 January 2013

Download the image sequence

Structure Sample Download sample
sample.zip [12 Kb]

Snapshot

Tube description

Tube
label
Length
nm
Radius
nm
Thickness
Membrane
Sample
Volume
Relative
Density
Approximative
surface
First Point (X,Y,Z)
nm
Last Point (X,Y,Z)
nm
Tube-033287.281.00.5 nm78431 nm31.066575 nm2 (34412.21, 18657.32, 100.00) (3987.79, 18657.32, 100.00)
Tube-130374.751.00.5 nm71569 nm31.060749 nm2 (34226.73, 18994.95, 100.00) (4173.27, 18994.95, 100.00)
Tube-230374.751.00.5 nm71569 nm31.060749 nm2 (34226.73, 19405.05, 100.00) (4173.27, 19405.05, 100.00)
Tube-333287.281.00.5 nm78431 nm31.066575 nm2 (34412.21, 19742.68, 100.00) (3987.79, 19742.68, 100.00)
Total127324.05300000 nm30.2546 μm2

Views of the sample

image

image

Fluorophores Download location
fluorophores.zip [23 Kb]

Parameters

Position
Sampling step on the tube axis0.250nm
Dispersion in X4068...34402nm
Dispersion in Y12861...25534nm
Dispersion in Z99...101nm
Experiment
Duration time2.00seconds
Intergration time20.00ms
Framerate50.00Hz
Number of frames1...41frames
Emission of photons
Extinction coefficient (EC)60000M-1.cm-1
Excitation wavelength702nm
Quantum yield1
Maximum laser power200W/cm2
Center of excitation X19200nm
Center of excitation Y19200nm
Non-uniform bumped excitationfalse
Flux of photons70.631020 photons/cm2/s.
Flux of photons per seconds97220.38photons / mol.
Flux of photons per frame1944.41photons / mol.

Activated fluorophores

Counting
Number of positions300molecules
Number of activations300molecules
Average number of fluorophores7.27mol./frame
Rate of activation1.00mol./frame
Density
Volume of the sample0.0003μm3
Volumetric density24227.62molecule/μm3
Field of view (image)1474.5600μm2
Imaging density0.0049mol./μm2/frame
Approximative surface of the sample0.2546μm2
Sample surface density28.5425mol./μm2/frame
Lifetime
Time profile of emissionConstant
Activation periodUniform
DelayFixed Value0.0 ms
AliveFixed Value40.0 ms
Number of photonsFixed Value
Point-Spread Function Download PSF
psf.zip [1162 Kb]

Parameters

Physical Model
Wavelength723.00nm
Numerical aperture (NA)1.40
Diffraction limit258.21nm
Offset focal plane 0nm
XY functionGaussian
FWHM258.21nm
Z functionExponential
Focal plane1 x FWHM at 0.0 nm
Defocus plane2 x FWHM at 500.0 nm
Computational Parameters
Pixelsize to PSF convolution5.0nm
Depth of the PSF300.0nm

Orthogonal Section of the PSF

Resolution: 5.0 nm per voxel

XY orthogonal section at focal plane z:0.0 nm

XZ orthogonal section at y:1030.0 nm

XY dimension: 2070.00x2070.00 nm

XZ dimension: 2070.00x300.00 nm

Reference

[1] H. Kirshner, F. Aguet, D. Sage, M. Unser, "3-D PSF Fitting for Fluorescence Microscopy: Implementation and Localization Applications", Journal of Microscopy, in press.

[2] PSF Generator An ImageJ plugin to generate 3D microscope Point-Spread Function (PSF).

Sequence Download frames
sequence.zip [2817 Kb]

Parameters

Camera
Photon converter factor or Quantum efficiency (QE)1.00e-/Ph.
QE = QE-Gain x QE-interacting [Ref. 1]
Resolution256pixels
Pixelsize150.00nm
Field of view38400.00nm
Optics
Wavelength723.00nm
Numerical aperture (NA)1.40
Diffraction limit258.21nm
Drift
DeriveDisable
ShakingDisable
Autofluorescence
Constant backround level (Mean)0.00
Constant background level (Standard Deviation)0.00
Location of the sourcesNone
Noise
Read-out noiseEnable
   ‐ Distribution: Gaussian (stdev)20.0
Dark noiseEnable
   ‐ Distribution: Poisson1.0
Shot noiseEnable
   ‐ Distribution: Poisson2.0
EM GainDisable
Hot pixelDisable
Dead pixelDisable
Analog Digital Conversion
Electron conversion - Gain1.00DN/e-
Electron conversion - Offset0.00DN
Baseline100.00DN
Saturation16383.00DN
Quantization14-bit
Computational Parameters
Number of activated fluorophores300
Thickness300.00nm
Frames (interval:1)From 1 to 41
MultithreadingOff-1 Thread
Verbose modeMute
Pixelsize to PSF convolution5.0nm
Pixelsize for adding autofluorescence75.0nm
Pixelsize of the camera150.0nm
File formatTIFF 16-bits
Fluorophores SNR
Min ..MaxMean
Peak signal s808.00..1696.00990.42
Background sdev σb17.50..341.8529.86
SNR (s-b)/σb2.73..64.3036.50
PSNR s/σb3.36..68.5640.73
SNR_sb (s-b)/sqrt(σb2s2)2.19..4.243.52

Reference

[1] James R. Janesick, Photon Transfer, SPIE Press Monograph Vol. PM170, 2007.

Excerpt

First Frame: 00001.tif

Download original image: 00001.tif

Random Frame: 00033.tif

Download original image: 00033.tif

Random Frame: 00025.tif

Download original image: 00025.tif

Last Frame: 00041.tif

Download original image: 00041.tif

Oracle Download oracle
oracle.zip [15 Mb]

Time projection

Average Intensity Projection

Camera Resolution: 150.0nm/pixel (Original size)

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High Resolution: 5.0nm/pixel (Enlarge this image)

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Maximum Intensity Projection

Camera Resolution: 150.0nm/pixel (Original size)

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High Resolution: 5.0nm/pixel (Enlarge this image)

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© 2017 Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
Last update: 31 Mar 2017