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Dear Pascal,

This is a known limitation of all structured illumination methods, including Apotome. Structured illumination uses a grid illumination to encode the depth of the structures in the specimen relative to the focal plane: if a grid projection on a structure is sharp, the object is known to be in the focal plane. If it's blurred, it lies outside the focal plane and is removed from the final image. In case of a homogeneous "sea of fluorescence" (such as Chroma slides), the algorithm cannot distinguish well between the blurred out-of-focus signal and the "real" blur resulting from the homogeneous fluorophore distribution, and therefore generates images with arbitrary levels of background. This results in a tile-scans with varying level of signal in different tiles that you observed. 

As a guideline for Apotome processing, only samples should be used that demonstrate a visible Apotome illumination grid (either in Live-Mode on the screen, or through the Oculars). Shading-correction should be done in wide-field mode. 

If you want to make sure that the Apotome processing works fine with a tile-scan, you could test it e.g. on a sample with a layer of fluorescent beads (that have some space between them) instead of your homogeneous fluorescent slide. 

I hope this helps!

With best regards,
Uros Krzic from ZEISS


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Pascal Lorentz
Sent: 06 November 2017 11:27
To: [log in to unmask]
Subject: Apotome tile scan

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Dear list

Not exactly a confocal question but I am sure I can get some feedback to my problem here:

We observed a strange behavior when using our Zeiss Apotome system for tile scanning. We acquired a 3x3 tile and can clearly see that some of the images are brighter than others (I used a chroma plastic slide here). A single image really pops out. If I draw a line profile over the three images in a row I can see a jump in intensity from one image to the next.
If I acquire the same tile again with the exact settings the pattern changes. So this time another image pops out or if we are lucky they all have the same intensity.
Do you have any idea why this happens? If we run a tile scan in wide field with the Apotome removed and the correct shading correction in place the line profile is perfectly flat.
So I assume there is an issue with the Apotome but I am not sure if this is hardware or software related. I am also not exactly sure how to properly correct for shading. As far as I understood we should use a shading correction acquired in wide field mode and use this during Apotome acquisition.
I would be very glad to hear from other people if they observed similar things with their Apotome and if there is a solution to this problem.

Thanks a lot and best regards

Pascal