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In microscopy high numerical apertures are desirable to capture as much light as possible from a small sample. A high numerical aperture (equivalent to a low f number) gives a very shallow depth of field. Higher magnification objective lenses generally have shallower depth of field; a 100× objective lens with a numerical aperture of around has a depth of field of approximately 1 μm . When observing a sample directly the limitations of the shallow depth of field are easy to circumvent by focusing up and down through the sample; to effectively present microscopy data of a complex 3D structure in 2D, focus stacking is a very useful technique.

Excelently explained! Thanks for this article.
Perhaps I should mention another stacking program which lets you mark certain points that one needs to co-ordinate and align throughout the stack.
This one is a standalone, so it can be used without Photoshop (for people who use other photo-editing software).
It is called RegiStax, current version update is - requires (6 may 2011). You need to have RegiStax 6 installed before installing this update.
It is freeware, and although intended for astronomy, it will work with all kinds of images. Find all the versions here:
http:///registax/

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