Seitenhistorie

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You can have more control if you extract the interesting portion of the image to a new image by using the Intelligent Cropper, and the ask for the statistics of that independent chunk (Edit > Statistics): you will obtain among other things the average value. Still, the cropper only allows you to crop or extract regions of cuboid shape.

But the ROI selection in the Object Analyzer can be used for more complex shapes. When you have a ROI defined anywhere in the image and click the "Analyze all" button, also information about the ROI is printed on the table, among others (by default, but you can change this) the sum intensity and the number of voxels. By dividing these two figures you can easily get an average value for the intensity inside the ROI.

Because defining a ROI in the background where not objects are normally found may by tricky, you may want to activate in the MIP pipe the same channel you are analyzing, so that you have a visual reference on where to define the ROI.

Background Mode

Background Mode is a Restoration Parameter in the Huygens Software. It establishes how the BackGround of an image is estimated in a deconvolution procedure.

The following choices are possible here:

• Lowest value (default): The image is searched for a 3D region with the lowest average value. The axial size of the region is around 0.3 micron; the lateral size is controlled by the radius parameter which is default set to 0.5 micron.
• In/near object : The neighborhood around the voxel with the highest value is searched for a planar region with the lowest average value.
• Widefield (WF) : First the image is searched for a 3D region with the lowest values to ensure that the region with the least amount of blur contributions is found. Subsequently the background is determined by searching this region for the planar region with radius r that has the lowest value.

The size of the region where the background is looked for can be controlled by the radius parameter in the estimate background tool, but for the deconvolution commands a default value is used.

If background mode is lowest, object or wf a background value will be estimated for each channel and frame in the image. In the Batch Script, with the mode set to auto the most appropriate method for the image is selected.

The estimated values can be modified by a percentage as defined by Background Per Channel.

If the background mode is manual then the value in Background Per Channel is taken as an absolute value, and will be removed.