Confocal Microscopy List - Re: Configuration problem with Zeiss LSM 510

Re: Configuration problem with Zeiss LSM 510

Posted by yee z on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Configuration-problem-with-Zeiss-LSM-510-tp2303218p2304260.html

Dear lister,

Thanks a lot for the feedback on my question.
From what you guys said, now I know I need to do pinhole adjustment for every new configuration. Learn something new everyday :-)

And I have another question regarding the pinhole adjustment.
Jean-Christophe suggested to use a mirror slide while Xuejun suggested to use Zeiss plant cross section slide, which one is better for this purpose?
I have a cross cut plant seed slice, can I use this one?

Thanks again!

--- On Tue, 2/10/09, Carl Boswell <[hidden email]> wrote:

From: Carl Boswell <[hidden email]>
Subject: Re: Configuration problem with Zeiss LSM 510
To: [hidden email]
Date: Tuesday, February 10, 2009, 12:16 PM

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: [hidden email]

To: [hidden email]

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 <[hidden email]> 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