Dear Aryeh,

Could you provide more information on your problem:
Is the non-uniformity you speak of, a bright signal in the centre of the
field of view, and darker at the edges of the screen? If not could you
describe the pattern of non-uniformity.

What lens are you using? Is it a Plan or Plan Apo? Dry, oil, water
immersion?

Are you "looking at the same wavelengths in each of the PMTs? This would be
quite difficult given the fixed dichroic for PMT3.

I think what you may be seeing is chromatic aberration. If you use a Plan
Apo lens the non-uniformity across the screen should be reduced. A zoom of
1.5 or 2 should ensure that you use the flattest part of your objective and
produce flatter optical sections.

The effect of chromatic aberration by it's very nature must be different if
you are looking at different wavelengths. Presumably you are splitting the
fluorescent signal using the standard A2 block (565LP) so green fluorescence
will be reflected to PMT-1 , orange-red to PMT-2 the MRC-1024 has a fixed
dichroic (640 short pass) so that red fluorescence is reflected to PMT-3.

These different wavelengths must be effected by chromatic aberration by
differing degrees. Unfortunately, where chromatic aberration is a problem
the signals in the three detectors should not match in Z. This is "normal".
You need to choose a lens system that minimises this aberration, ie. Plan
Apo and minimise Refractive Index (RI) mismatch. This may be difficult if
the RI of your plastic is unknown, although imaging very close to the
surface of your block should help.

If you need to repeat the test I suggest that you would need to place beam
splitter block in and compare PMT-1 to PMT-2 with the open position chosen
for the band pass filter. A suitable band pass filter would then have to be
screwed into the rear of you first filter cube. This way PMT-1 and PMT-2
will "see" the same colour, except that you still have the 640SP infront of
PMT-1. To circumvent this problem the band pass filter you placed in the
first filter cube could exclude red wavelengths. Comparison of PMT-3 under
such controlled conditions is more difficult as the dichroic is fixed, and
so is probably not worth trying.


Stephen H. Cody,
Colon Molecular and Cell Biology Laboratory,
Ludwig Institute for Cancer Research,
Post Office Royal Melbourne Hospital,
Parkville, Victoria 3055, Australia.

Tel: 61 3 9341 3155   Fax: 61 3 9341 3104
email: [log in to unmask]

> ----------
> From:         Aryeh Weiss[SMTP:[log in to unmask]]
> Reply To:     Confocal Microscopy List
> Sent:         Friday, July 16, 1999 3:22 AM
> To:   [log in to unmask]
> Subject:      Field uniformity on an MRC-1024
>
> I found that when imaging a uniform fluorescent block, the resulting image
> is
> not uniform (even well beyond the acceptance criterion). Even after
> allignment
> of the
> mirrors, there can be a variation of as much as 50 grey level across the
> field
> at zoom=1.
>
> What is worse -- the variation across PMT3 do not track the variations
> across
> PMT1 and PMT2. The variation across PMT1 and PMT2 track reasonably
> closely, but
> not perfectly.
> This means that it is not just nonuniform illumination.
> It also means that comparing relative strengths between channels is
> difficult,
> and in the case of PMT3, probably not possible at zoom=1.
>
> For higher zooms, the results are of course better, but even at zoom=2
> PMT3 is
> not great.
>
> Is there any experience out there -- particularly among MRC-1024 users --
> that
> may
> be useful for me on this problem?
>
> Thanks in advance
> --aryeh
> --
> Aryeh Weiss                          | email: [log in to unmask]
> Department of Electronics            | URL:
> http://optics.jct.ac.il/~aryeh
> Jerusalem College of Technology      | phone: 972-2-6751146
> POB 16031                            | FAX:   972-2-6751275
> Jerusalem, Israel                    | ham radio: 4X1PB/KA1PB
>