Two points come to mind:
1.  The microscope may use the eyepieces to correct aberrations of the objective, in which case by removing them from the system the aberrations will not be corrected.  This is quite common, though lateral chromatic aberration is usually the problem here.
2.  The magnification is not conclusive proof - 1:1 transfer lenses are quite routine.  If there is no transfer lens the CCD chip must be the same distance from the objective as the base of the eyepieces - this should be susceptible to a visual check.  

But in any case the cause should easily be determined by looking through the eyepieces!  If the edges are in focus at the same plane as the centre the fault must be in your camera setup.  A very simple high-contrast test specimen (invented by Ron Oldfield) is to lightly (very lightly) smoke a coverslip over a match.  Scratch this a few times to create clumps for easy focussing.  Put it on a slide with a drop of immersion oil.  This is an amazingly effective test specimen for a whole lot of aberrations.  Lateral and axial chromatic aberration, field curvature and astigmatism all stand out like the proverbial canine appurtenances.

                                                      Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of David lenzi
Sent: Friday, 1 June 2012 5:23 PM
To: [log in to unmask]
Subject: field flatness problem (wide field)

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Thanks to
everyone who replied to my filed-flatness post with very helpful ideas.  The suggested causes included: 
-       Hitting the design limit of the objective due to the big camera chip
-       Additional lens elements in the light path that are not properly flat field corrected
-       Damaged or mis-seated objective
-       Incorrect coverglass
-       Sample too thick or too distant from the coverglass
-       Incorrect immersion oil

To give some additional info:
There are no additional lens elements between the tube lens and camera, confirmed by the pixel size which I measured at 0.1031 um, which is as expected for the 6.5 um pixels at 63x.

I am using Schott high tolerance coverglass (170 um +/- 5 um), and the specimens are 100 nm ultrathin sections (as for EM), adhered tightly to the surface of the coverglass.  So the glass and samples should be exactly appropriate for the objective. 

 I have so far used only the Leica immersion oil, but I think it is working well since point-spread functions of 100 nm beads come out at about 1.3x theoretical, indicating that we are imaging at close to the diffraction resolution limit.  At least at the center of the field!  I do have a selection of Cargill oils I could try though.

I don’t think the objective is damaged or mis-seated, but I’ll re-check, as well as try to swap out parts if possible, or borrow another stand.

As for hitting the design limit of the objective, that is a strong possibility, but I am waiting, patiently, for Leica to get back to me on this.

Finally, how have others mounted these big-chip cameras?  When the camera was delivered, it had a C-mount, which was clearly causing vignetting.  We got Fairchild to change it to an F-mount, whose bigger bore removed visible vignetting.  However, could the mount still be blocking some of the light (a diffraction effect), causing the focus issue?  I could see this changing resolution rather than focus, but I nevertheless plan to try removing the F-mount and putting the camera on a lab-jack as a test, as suggested. 

Thanks a ton for all the help,   -David

David Lenzi  PhD
Aratome LLC
Menlo Park CA, USA
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