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Running ZOLA PSF localization on test dataset

We provide instructions on how to run 3D localizations on the test data from the ZOLA package.

Test data

Download and unzip TEST-DATA.zip to get Mitochondria test data.

Mitochondrial protein TOM22 immunolabelled with Alexa 647 with corresponding bead calibration stack is imaged using tetrapod PSF.

Localization

  1. Open Mitochondria-tetrapod/data/Mito-sp4-SR-data-crop-small.tif
  2. Select Plugins -> ZOLA -> Localization and enter optical and computational parameters. Click OK to start localization.

localization dialog

  1. You will soon see automatically updated color-coded histogram.

automatic histogram

  1. Once localization is done (elapsed time ~4 minutes on our Tesla GPU), you can filter the localization table. Select Plugins -> ZOLA -> Filtering. You will see three new windows: disclaimer, scatter plot which can be used to select ROI, and brightness and contrast dialog, which represents now histogram on z localizations. We select a broad peak corresponding to the useful data and avoiding artifacts in the lower part of the axial range. Click OK, enter maximum value of Chi2 = 3 and click OK once again. Now our table is filtered.

filtering dialog

  1. In order to an axial projection we need to generate 3D histogram Plugins -> ZOLA -> Visualization -> 2D/3D historgam

histogram dialog

  1. Next, run Reslice (keyboard shortcut '/') form the left to see YZ stack. Mitochondrial organelles' hollow structure is readily apparent.

mito ZY projection

  1. Don't forget to save updated localization table Plugins -> ZOLA -> Import/Export -> Export table