How far above and below the in-focus region should I sample?
This question can be particularly relevant for widefield microscopy. Widefield images are fuzzy in the neighborhood of the in-focus region, which rises the question of how far above and below the in-focus region (Z direction) the image should be recorded to ensure an optimal deconvolution result.
For a good widefield deconvolution it is not necessary to acquire large regions above and below the in-focus zone. In widefield microscopy the blur can spread through large regions. The Huygens algorithms take this into account, thus imaging very far above and below the in-focus region is not necessary to achieve good deconvolution results. The image can be cropped far from the in-focus region to speed up the deconvolution process. To avoid cropping slices that contain useful information, leave a border of around 1-2 Half Intensity Width sizes. As an example, in widefield, well-sampled images recorded with NA 1.3 this corresponds to 0.75 to 1.5 microns.
A confocal image deconvolution already benefits from including a few planes. Even including one neighboring plane on each side of the plane of interest gives already an improvement in resolution and signal. However, if a realistic representation of an object in the plane of interest is required, it is advised to image at least a Z-stack that matches the dimensions of the PSF. You can calculate this with the calculator on NyquistCalculator. For larger objects you would want to have at least a number of Z planes that matches half the axial width of the PSF on both sides of the object.
Keywords: extra slices outside object
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs
Platforms: Irix Linux Windows Mac AIX
Related products: Hu Ess Hu Pro
