Background estimation

The background in your image is information you want to remove in the final result. It is normally composed of many constant signals, plus non-specific stainings all over the sample which are affected by Photon Noise and therefore are not constant at all. (See BackGround for more details).

You may be also interested in considering some low intensity regions in the image as background, in order to enhance high intensity objects.

Background estimation is part of the deconvolution procedure. The deconvolution Wizard in Huygens Essential and Huygens Professional clearly asks the user for the preferred approach. This mode of background estimation can also be set under deconvolution parameters in the Professional Operation Window or the Batch Processor template. Depending on the selected Background Mode, the averaged background level will be estimated in a region far from or near the main objects in the image. There is also a special mode intended for widefield images.

This estimated value must be compared with what you actually consider background in the image. Open the image with the Twin Slicer and look for a region that you want to remove. Move the mouse over it and see if the averaged value is representative of that region. If not, you can correct it.

After the estimation you can indicate a percentage of correction to be applied to the automatic value. For example, set this to zero to accept the estimated value as is, or set it to -10 to reduce the estimated value in 10%. If you manually modify the background absolute value, the change will also be reflected in the percentage correction.

A normal deconvolution procedure usually requires tuning the background value until the results fit your experimental necessities. As you can restart the deconvolution to use different parameters, you can test and compare which is the best background value. When you find it, you can apply it (in absolute terms, or as a percentage of correction to the automatically averaged one) to all your images.

A deconvolution example where the background is tuned until it fulfills the user's experimental requirements is detailed in Tuning Huygens Deconvolution. In this example, Huygens Professional is used instead of Huygens Essential. That is why some Tcl Huygens commands are shown as reference. But don't pay attention to them if you don't want to, what matters is the idea that tuning the background can take you to the results you want.

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