Background of a 3D image

Background is any additive and approximately constant signal in your image that is not coming from the objects you are interested in. Background might come from for example an electronic offset in your detector or from indirect light.

The extended (and not enough considered) assumption in imaging is that the digital microscope reports intensity levels of light which is directly related to the local density of molecules in the preparation. If one pixel reads 100 (in whatever units) and another reads 25 , we interpret that the first one 'contains' four times more concentration of fluorophore than the second pixel. If you have an offset (let's say +30) affecting your readout (because the whole thing is soaked in non-specific staining, for example) and intensities are shifted to 130 and 55, the ratio 4/1 is lost, and your intensity interpretation is compromised. That, independently of whether you deconvolve the image or not, may only enhance the mess...

Generalities

The mean background in an image in general is composed of (electronic) offset, scattered light, and light from a-specific labeled components. In most cases the electronics-induced offset will give rise to a noise-free background, also called the baseline or Black Level. The baseline can be found by recording a black image. The other components will be subject to Poisson noise due to the strongly photon limited character at typical background light levels.

Therefore, the contribution to the background of these last components will show up in the recorded image as background noise composed of randomly scattered bright pixels or VoXels against a dark homogeneous background (the baseline). The background may be affected by Bleaching Effects due to the a-specific labeled components; the baseline component obviously isn't.

In the case of Wide Field images or confocal Time Series the restoration tools in the Huygens Software will automatically attempt to correct for bleaching since this is vital for effective blur removal (see Bleaching Mode). However, if the Black Level component is large with respect to 'real' image data automatic bleaching correction will fail. (This black level is normally non-existing in CCD cameras, but sometimes a current bias is used). Because it is hard for the software to distinguish between the different background components, something vital for the flux equalization in widefield images, we advice to subtract the baseline, if any is present, prior to any further processing. This can be done with the Adjust baseline tool or with the arithmetic functions in the Huygens Professional.

The background component due to the a-specific labeled components will in general be rather noisy. Without the help of the Huygens Software's restoration tools it is very hard to remove this background noise without degrading also the objects in the image. Fortunately, all restoration tools contained in the Huygens are able to remove the noisy background without degrading the image. To do so, they need the value of the mean fluorescent background: the average background in a non-object area in the image.

Background mode

Sometimes there is need for defining the background inside an object, for example a cell. The option in object is available for that purpose. For widefield images, the widefield mode is recommended. See Background Mode for more details.

The background parameter

The background parameter used in Doing Deconvolution by the Huygens Software can be stablished in absolute terms, on relatively to the automatically estimated background level.

Using relative values (in percentages) to correct the background estimations is a good procedure to apply comparable background corrections to a series of images. And in all cases the user has the control on what is going on during the Image Restoration: it is the user who specifies what is background to be removed and what is not.

Estimating the background

In Huygens Essential estimating the background is part of the deconvolution procedure. 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 be also reflected in the percentage correction.

For more complex ways in Huygens Professional, see Background Estimation.

In the Professional and Scripting deconvolution commands

In the Huygens Professional and Huygens Scripting, the automatically estimated values can be modified when doing deconvolution by a percentage as defined by the Background Per Channel parameters.