Here's some potentially unconventional suggestions for you, as I don't know the resolution needed for your client's story to be told.  However, if you'd like to get more light through the SDC system, take away some of the elements that are preventing light from travelling through the optics.  You may not need to perfectly match the pinhole size with your objective to gather the essential data from your live cell imaging experiment.  How about trying a lower power objective with less glass elements in it?  You will effectively gain some useable signal with each of those choices, while potentially losing some via NA, depending on the objectives in your quiver.

Perhaps if you shared with the list what the client was trying to image, then perhaps that might yield more insights.

Cheers,

--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office (901) 495-2536
Cell (901) 603-3162
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On 11/24/08 5:08 PM, "Mayandi Sivaguru" <[log in to unmask]> wrote:


Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv




Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: 217.244.2496
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http://core.igb.uiuc.edu

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