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Hello Leoncio,

Have you thought of trying Turbo Sapphire FP for labeling histone. Since you already are restrained by your other tags this should be ok. We used it a few years ago in combination with other FPs on our spinning disc. It seem to work well enough for what we needed. Also, I am just curious can use DIC on your swept field?

Hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Vergara, Leoncio A.
Sent: Monday, July 25, 2011 4:20 PM
To: [log in to unmask]
Subject: Long term Nuclear labeling in live cells

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I was wondering if there is any non toxic alternative help in segmentation of nucleus and cytosol compartments to measure protein translocation dynamics in 12-24 hrs time lapse experiments in live cultured cells. 

We are working with cell lines stably expressing both GFP and Strawberry fluorescent protein constructs. We want to follow the single cell translocation process and correlate the two signals over a period of 12-24 hrs. We are looking for a tool to aid in segmentation between both compartments for automated image analysis. The problem is that all the DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI, HOESCHT and several SITO dyes from molecular probes. We have not tried the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be compatible with GFP and Strawberry, but a call to their technical support was not very encouraging. 

I am wondering if there are any long term live cell tracking dyes that can label the cytosol and give a "negative" image of the nucleus, be non toxic and be compatible with GFP and Strawberry.

Invitrogen also has the CellLight reagents but they are base either on GFP or RFP so won't be a solution either since we are using those channels. I guess we could develop a far-red construct for triple FP labeling, but I was hoping for an easier solution... :)

For additional reference: the microscope we are using is a Prairie Technologies Swept Field Confocal, we have 4 channels available (typical DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.  

Thanks in advance, any suggestion would be greatly appreciated

Leoncio Vergara
Technical Director
Optical Microscopy Core
Galveston 
Texas