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G'day Sarah,
For confocal microscopy I would definitely recommend the SNARF dyes. They are emission ratiometric, which means that if you co a calibration curve the data is a true quantitative measure of pH, that is independent of dye distribution and photobleaching artefacts. Unlike Calcium dyes the calibration process is quite straight forward.
See papers:
Cody, S.H., Dubbin, P.N., Beischer, A., Duncan, N.D., Hill, J., Kaye, A., & Williams, D.A. Intracellular pH mapping with Snarf-1 and confocal microscopy. I: A quantitative technique for living cells and isolated tissues. Micron 24(6): 573-580, (1993). http://dx.doi.org/10.1016/0968-4328(93)90034-X <http://dx.doi.org/10.1016/0968-4328(93)90034-X>
Dubbin, P.N., Cody, S.H. & Williams, D.A. Intracellular pH mapping with Snarf-1 and confocal microscopy. II: pH gradients within single cultured cells. Micron 24(6): 581-586, (1993). http://dx.doi.org/10.1016/0968-4328(93)90035-Y <http://dx.doi.org/10.1016/0968-4328(93)90035-Y>
Cannell, M.B. and CODY, S.H. Fluorescent ion measurement. In: Handbook of Biological Confocal Microscopy. 3rd. Edition. (Ed. James Pawley). Chapter 42 (2006)
Cheers
Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
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Parkville, Victoria, 3050
Australia
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________________________________
From: Confocal Microscopy List on behalf of Sarah Tanton
Sent: Fri 06/07/2007 1:08 AM
To: [log in to unmask]
Subject: pH indicators
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear List,
I have been tasked to image changes/movement/presence etc. in the pH around neural growth cones. I am considering using BCECF and SNAF dies could anyone make a recommendation of one over the other?
Thank you for your time,
Sarah Braun
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