Hi
Yes the 'pinhole not adjusted' warning always appears if you create a 'new'
optical filter configuration. Although you can manually adjust the confocal
pinhole [even incorrectly] to kill off the nagging & annoying message on the
LMS510, Zeiss do provide a utility to do this automatically for you for all
the filter configurations you have.
You do this while focused on the ‘mirrored’ slide Zeiss kindly gave you with
the confocal when you bought it, and you run a little program that
automatically aligns all the pinholes for all the optical configurations you
use [even all those that don't display the warning message]. You switch on
the 633nm laser select the 10x objective [always]. Leave the system to warm
up for at least a couple of hours. Put the mirrored slide under the 10x and
focus on it – normally you can see dust which helps or use the palettes to
show over exposure [red] and when it’s brightest you’ve hit the mirror.
You then run PHAdjust.exe from Zeiss’s AIM folder [C:\aim\hwt\PHAdjust.exe
on our PC]. This will automatically adjust all the available beam paths in
your filter config files. For any other users with unusual filter
configurations just ‘resuse’ the beam path from one of their images and save
it in your configs.
Generally I run PHAjust.exe. every month. It takes about an hour to
auto-adjust all the optics correctly and as a bonus gets rid of that
annoying message.
Regards
Keith
---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford OX3 7BN,
United Kingdom.
Telephone: +44 (0)1865 287568
Email: [log in to unmask]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
________________________________________
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Carl Boswell
Sent: 10 February 2009 17:17
To: [log in to unmask]
Subject: Re: Configuration problem with Zeiss LSM 510
All I can add to what Estaban said was be sure to Save the position after
you have made the adjustments. Adjust the image Gain using the
Palatte>Range Indicator so there is some saturation in it so that you have
an objective way to see if your changes are making things brighter or
dimmer. Adjusting the columator may also improve your image.
Carl
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
----- Original Message -----
From: G. Esteban Fernandez
To: [log in to unmask]
Sent: Tuesday, February 10, 2009 9:12 AM
Subject: Re: Configuration problem with Zeiss LSM 510
You need to go into the Maintain > Pinhole menu and optimize the X/Y
positions of pinhole 3. The message means that has never been done for that
particular configuration of dichroics. The pinhole is probably not centered
directly in front of the PMT so you don't get a good image if it is closed
down, but you'll probably get an image if you open the pinhole to max.
-Esteban
On Tue, Feb 10, 2009 at 9:54 AM, yee z <[log in to unmask]> wrote:
Dear listers,
We have a Zeiss LSM510 confocal microscopy system at our core facility. And
I got some problems while writing new configuration.
Here is an example:
Single track configuartion for FITC, I used to use HFT 405/488/543/633, NFT
545, NFT 490 and BP505-550. It works fine. (Screen shot:
http://i561.photobucket.com/albums/ss60/mizhzmkm/Q-1A.jpg)
But when I try to change the primary dichroic beam splitter to HFT 488/594
and keep everything else the same, the systme shows "Pinhole PH3 is not
adjusted". (Screen shot:
http://i561.photobucket.com/albums/ss60/mizhzmkm/Q-1B.jpg)
Also if I change the NFT 545 to NFT 595, the system also shows "Pinhole PH3
is not adjusted" (Screen shot:
http://i561.photobucket.com/albums/ss60/mizhzmkm/Q-1C.jpg)
I am still be able to take the images regardless the error window.
So does anyone here have the same problem? What does "Pinhole PH3 is not
adjusted" mean, any reason for this?
Any suggestion will be appreciated.
Thanks,
Yi
--
G. Esteban Fernandez, Ph.D.
Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO 65211
http://www.biotech.missouri.edu/mcc/
573-882-4895
573-884-9395 fax
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