Batch mode

Small Fish comes whith a batch mode allowing you to leave your computer process a batch of images on his own at the cost of interactivity with quantification(napari features). Before starting batch mode, place all your images with appropriate format, alone, in a folder. Also it is recommended to have quantified a few images to beforehand to fine tune your parameters.

Get started with batch mode

To begin utilizing batch mode click Batch detection in main menu.

First click on the Browse button on top of the window and select your folder then click the load button situated next to the Browse button. A list of all files found in the folder will be displayed, when launching the analysis, Small Fish will process one by one all these image files. To ensure it will be able to run from start to finish without running in an issue you may click the Check button and a coherence check will be perfomed between your images (time consuming for large batch).

Next step is to configure pipeline, all configuration are equivalent to classic use of Small Fish excepted for all options that use napari. Also note that to perform segmentation the option Cell segmentation must be ticked in the Input tab. To access Segmentation and Detection parameters tabs the Input and Map tabs must be filled correctly. Finally, let's have a look at the Output tab :

Click the Browse button to select a location where your analysis folder will be created. Then enter the name for this batch analysis and check the options for quantification visualisation you would like :

• save segmentation : Saves png image files whith segmentation borders for cytoplasm and nucleus. Note they are not actual mask to be load in python (.npy) they are only meant for you to evalute your segmentation quality.

• create spot detection visuals : (hard drive memory intensive) This will create addition .tiff files meant to be open in ImageJ or Napari, where first channel is raw signal from your fish (channel selected for detection), second channel is fake signal whith only detected spot and last channel is fake signal with only detected clusters (if clustering performed). Again this is meant only for you to assess detection quality.

• extract spot : For each image, extract for each spot coordinates, intensity, cell information etc... This is a deeper information level than cell level. For more information see Quantification section.

When all settings are correctly set Start will higligh, click it to launch analysis ! During analysis you will not be able to interact with the software, to close and cancel quantification, close small fish terminal or press Ctrl + C in terminal.