Step by step Guide for eC-CLEM AutoFinder Totally automatic Registration: Example with visual protocols

Download the data sets and look for automatic registration in Icy This will open a visual protocol

This example will process only one pair of images, but extending it to batch processing is straight forward http://icy.bioimageanalysis.org/plug in/Protocols

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Input needed 1.

1. Source Image: 2014-1211_Bloc1_G2_S4_20-8bits.tif

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2. Target image: Flat-Denoised8bits.tif

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3 and 4: directory where to save the resulting images (i.e target on source and source on target)

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5: press Run when Ready

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Advanced use: Visual protocol Autofinder can be called from a visual block, and be adapted to your own CLEM workflow 1.

1: Detect spot in source image

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2:Detect spot on target image

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3: Use Autofinder block

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Equivalent manual steps: Detect spot in source image (with Output/export to Roi checked)

Equivalent manual steps: Detect spot in target image (with Output/export to Roi checked). Use Channel 2 showing melanosomes for the detection

Equivalent manual steps: Detect spot in target image (with Output/export to Roi checked). Select the sacle and press Start Detection

Equivalent manual steps: Launch autoFinder

Results Due to the random aspect of the procedure optimization, the numbers kind be slithly different (here: on the 334 spots detected on source image, 143 spots over the total 646 spots in target images were in the candidate circle, and 100 (70 %) have been matched with a spot in the source image, with 0.36 micrometers as average distance, below the maximum 1 micrometers maximum distance given in parameter.