Coloc-TesselerInstallation guide & Manual

By F. Levet & JB. Sibarita

Team Quantitative Imaging of the Cell

Interdisciplinary Institute for Neuroscience

CNRS UMR5297, Univ. Bordeaux

Installation (1/2)

• Need a PC with Windows (7/10) and OpenGL installed

• Launch « Coloc_Tesseler_Setup.msi »

• Select installation directory

Installation (2/2)

• Installation folder contains executable, icons, fonts, DLLs files anddataset examples

• Unzip « Datasets.zip » example files (in Datasets directory). This willcreate three sub-directories (Experimental, Simulations_2D andSimulations_3D)

Coloc-Tesseler Quick start (1/3)

• Launch « Coloc-Tesseler.exe »

• 2 windows will pop-up• A Console (for application messages)

• A fullscreen Viewer

Coloc-Tesseler Quick start (2/3)

• Single-molecule data are in csv format (Thunderstorm)

• To open single-molecule data:• Click on the open icon

• Choose « Open Localization File » to open a single file

• Choose « Open Dir » to open all files in a given folder

• Choose « Open Dir »• Go to Coloc-Tesseler\Datasets• Single click on « Simulations_2D »• Click on « Choose »

Coloc-Tesseler Quick start (1/3)

• Six datasets are opened (corresponding to three 2-colors simulations)

Interaction panel

Coloc-Tesseler GUI – Misc. Tab (1/1)

• Click on the background color to change it

Coloc-Tesseler GUI – Localizations Tab (1/2)

• Single-molecule detections can be displayed by checking the« Display » check-boxes

Coloc-Tesseler GUI – Localizations Tab (2/2)

• Change parameters of the histogram by middle-cliking on it

• Click on the color button for changing the LUT

• Check the parameter you want to change and enter the new value

Coloc-Tesseler GUI – Toolbar (1/2)

• Zoom by clicking on the glass icon

• The zoom is continuous: hold the mouse clicked and move the mouseup or down to zoom-in or zoom-out

• Click on the hand to move laterally in the data

Coloc-Tesseler GUI – Toolbar (2/2)

• Click on the « Colocalization » Icon to bind the 2-colors of a dataset

• Check « All 2-colors datasets » to bind all the opened dataset by 2

• Check « One 2-colors datasets » to choose the 2 datasets to be bind

• Click on « Ok »

Coloc-Tesseler GUI (1/1)

• Three 2-colors datasets are created• First color is displayed in red

• Second color is displayed in green

• Voronoï diagrams for both colors are created

Coloc-Tesseler GUI – Colocalization Tab (1/4)

• Coloc-Tesseler automatically computes the Spearman/Manderscoefficients and classifies the localizations• At this stage, Coloc-Tesseler uses a threshold of 1

• Interaction is possible through the « Colocalization » tab

Voronoï scatterplot

Display options

Threshold options

Colocalization values

Coloc-Tesseler GUI – Colocalization Tab (2/4)

• (Un)check the corresponding check-boxes to (not) display theclassified localizations (Color A & B), the classification backgroundclass (black localizations), the original localizations and the Voronoïdiagrams

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2

3

1 2 3

1 + 2 + 3

Coloc-Tesseler GUI – Colocalization Tab (3/4)

• Check « log » for log display of the Voronoï scatterplot

• You can change the grid size of the color map

Coloc-Tesseler GUI – Colocalization Tab (4/4)

• Modification of the thresholds can be done in two ways1. Direct click inside the scatterplot

2. Direct modifications of the thresholds

• The use of one of these methods will update the other

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2

Coloc-Tesseler GUI – ROIs (1/2)

• Click on the « Define ROI » to create ROIs• Line, circle, square, triangle & polyline are possibles

• ROIs can be saved, loaded, discarded and are accessible in the ROI manager

Coloc-Tesseler GUI – ROIs (1/2)

• Colocalization analysis is automatically performed on ROIs

Coloc-Tesseler GUI (1/1)

• Click on the « Close All » to close all datasets

Coloc-Tesseler – Experimental dataset (1/1)

• Open directory « Coloc-Tesseler\Datasets\Experimental »

• Click on the « Colocalization » Icon with « All 2-colors Datasets » checked

• Load ROI file « spinesROIs.txt » for colocalization analysis on selectedsynapses

Coloc-Tesseler – 3D simulations (1/2)

• Open directory « Coloc-Tesseler\Datasets\Experimental »

• Four localization files are opened (two 2-colors datasets)• 1st condition: Full colocalization (distance between 2-colors clusters is 0 nm)

• 2nd condition: Partial colocalization (distance between 2-colors clusters is 50 nm)

Coloc-Tesseler – 3D simulations (2/2)

• 3D Voronoï diagrams are projected on a 2D camera• Difficult to understand uncheck the Voronoï diagrams rendering

By being 2D, ROIs shouldnot be used with 3D

datasets