Our Zeiss does not change the pinhole size -ever. Sounds to me like a
bad electrical connection to that pinhole motor... Can you check this?

Regards

Mark Cannell

On Mon, 24 Mar 1997 11:11:32 -0400 Xue Jun Sun wrote:

> From: Xue Jun Sun <[log in to unmask]>
> Date: Mon, 24 Mar 1997 11:11:32 -0400
> Subject: Pinhole size on Zeiss LSM 410
> To: [log in to unmask]
>
> Hi, Everyone,
>
> I am asking for help to other Zeiss LSM 410 users.
>
> Problem:
> It appears that the computer is adjusting the pinhole size
automatically
> (and unpredictably). When I was scanning a stack, the image would
appears
> suddenly brighter (or mostly dimmer). This can happen any time
during a
> scan. So either I obtain a uneven brightness single image or the
> brightness of the image is changed from one to other in the same
stack. I
> know it affects the pinhole because once, it puts itself into
non-confocal
> mode and back to confocal mode late on. This problem existed for
quite a
> while now. But it does not happen all the time. So it make really
hard to
> track down what is really going on. At beginning, we can hear the
small
> noise when you change from confocal to non-confocal mode when the
image
> brightness changes. Now this problem is still there even we do not
hear
> the noise.
>
> This problem happens only to one of the two pinholes.
>
> What I got from Zeiss representative (via local manager of the
facility,
> so it is not a quotation):
>
> 'I was setting pinhole too small (12 for an objective 25X, NA 0.8
Zoom
> setting 1, a setting gives roughly an airy disk of 1) and the
pinhole is
> too sensitive for that setting. So it fluctuates.'
>
> It is true set the pinhole bigger (20) seems help. But careful look
still
> shows the problem. Just less dramatic. I was not satisfied with this
> explanation because it did not explain why it did not happen with
another
> pinhole with the same setting. Moreover, should a pinhole size
change when
> it is setting too small?
>
> What I have tried.
> I know this problem is not related to 1) laser power; 2) pinhole
size
> setting, 3) interpolation on/off 4) scanning speed. What seems help
(but
> did not eliminate the problem) is when I set all detector same
pinhole
> size and coupled pinhole in the pinhole setting window.
>
> Any idea or suggestions? Please send the reply to the following
Email
> address if you think it is not generally interesting.
>
> Thanks in advance.
>                                 Xuejun Sun, Ph.D.
>
> ********************************************************************
> Xuejun Sun, Ph.D.
> Dept. Psychology
> Life Science Centre
> Dalhousie University
> Halifax NS B3H 4J1
> Canada
>
> Email:  [log in to unmask]
> Phone:  (902) 494 3746
> Fax:    (902) 494 6585