Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy

Computational algorithms for three-dimensional deconvolution have proven su ccessful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions a nd the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the a lgorithms. in this paper, artefacts associated with a decrease of fluoresce nce intensity with time or slice in three-dimensional wide-field images are demonstrated using simulated images. Loss of intensity, whether due to pho tobleaching or other factors, leads to artefacts in the form of bands or st ripes in the restored images. An empirical method for correcting the intens ity losses in wide-field images has been implemented and used to correct bi ological images. This method is based on fitting a decreasing function to t he slice intensity curve computed by summing all pixel values in each slice . The fitted curve is then used for the calculation of correction factors f or each slice.