Why do my deconvolved images look darker than the raw images?

The deconvolution process increases the Dynamic Range of the dataset, i.e the intensity range increases significantly..

So your SNR increases often 3 to even 10 times (for STED images for example) with as result that the dynamic range of your dataset increases accordingly at least threefold or much more.

A computer screen can not display arbitrarily high intensities, there is a maximum for the brightness of a pixel in the screen. Therefore the maximum intensity in an image is normally mapped to the maximum intensity a screen can display, and all the other pixels in the image are scaled accordingly.

Since the image is auto-scaled between the maximum and minimum intensity when displayed on the screen, the background features will appear darker. If you have a very bright point somewhere in the image, almost any other feature will appear very dim. It is as if you were dazzled by the bright feature.

So if you visualize them linearly in the Huygens Twin-slicer the low intensities look dimmer due to this increased range. You can let them come out better by opening them in the Huygens Twin Slicer (Advanced Mode) and selecting as projection mode MIP or SUM, plus selecting for the contrast mode for example COMPRESS or STRONG COMPRESS and if necessary using the Contrast Editor. For more information on how to use the Twin Slicer, see its chapter in the Essential or Professional manual which can be downloaded from here.

You can also check your Point Spread Function (PSF) to make sure that it is calculated based on the right parameters or distilled out of beads that were acquired in the same conditions as the image. If the PSF is too large, the deconvolution will remove many features that are interpreted as wrong objects. (Mind that anything in the image smaller than a PSF shouldn't be there in the restored image and will vanish: the PSF is the image of a single mathematical point, and nothing in the restored result can be smaller than that). You are advised to double-check the size of the PSF that you are using, and compare it with the size of the features in your image and with a theoretical PSF that you can manually calculate in Huygens Professional or using our Nyquist Calculator on-line tool.

So this dimming effect increases with the much higher SNR after deconvolution with Huygens

If there are very bright pixels of high intensity somewhere in your image, all the other pixels will look relatively dim. The brighter these pixels are, the dimmer will the other pixels look.

Due to the much improved SNR after the deconvolution you are also allowing more light to be gathered to their source pixels, increasing the deblurring effect of the restoration. This will of course change the relative intensity between bright and dim pixels, making the image look dimmer in most places.

Keywords: dynamic range intensity gamma dark image
Categories: Faq
Platforms: Linux Windows Mac
Related products: Hu Ess Hu Pro