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Dear Patrizia,
I have worked with confocal (not multiphoton) with thick specimens
measuring pH with SNARF-1/AM. This worked very well with whole isolated
rat muscles. I don't know if the SNARF range of dyes are suitable for
MPE though.
You should use a "Cold Loading" protocol to allow the dye to diffuse
throughout your tissue before being cleaved into the active form. So yes
with care you should have no problems with the thickness of the tissue.
eg. 4 Deg C for 60 minutes followed by post incubation at 37 Deg C for
30 minutes to cleave the dye.
You need to use more dye on tissue specimens than you would with
isolated cells, simply because of the large mass of tissue involved. If
you use too little dye there will not be enough SNARF molecules per cell
to gain enough signal. Too much dye and you could buffer any pH changes
you are attempting to measure. So you will need to play around with dye
concentrations.
See any or all of the following:
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
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
Williams, D.A., CODY, S.H. & Dubbin, P.N. Introducing and calibrating
probes in cells, organelles and membranes. In: Fluorescent and
luminescent probes for biological activity - A practical guide to
technology for quantitative real-time analysis (ed W.T. Mason) Academic
Press Limited, U.K. Chapter 25, pp 320-334, (1993).
Bowser, D.N., Cody, S.H. & Williams. P.N. Introducing and calibrating
probes in cells, organelles and membranes. In: Fluorescent and
luminescent probes for biological activity - A practical guide to
technology for quantitative real-time analysis (ed W.T. Mason) Academic
Press Limited, U.K. 2nd edition. Chapter 5 pp 65-81. (1999).
Cannell, M.B. and Cody, S.H. Fluorescent ion measurement. In: Handbook
of Biological Confocal Microscopy. (Ed. James Pawley) Springer, NY. 3rd
edition. Chapter 42 pp 736-745 (2006)
Cheers
Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Victoria, 3050
Australia
Tel: 61 3 9341 3155 Fax: 61 3 9341 3104
email: [log in to unmask]
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Patrizia Camelliti
Sent: Sunday, 30 March 2008 12:08 AM
To: [log in to unmask]
Subject: pH and oxygen gradients
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear all,
I'd like to measure pH gradients and/or oxygen gradients in living
tissue
slices about 350 micron thick. Does anybody know a good method? Is there
any dye I could use with a 2-photon (wavelenght 700-990)? Will the dye
be
able to reach the deep layers of cells in the slice?
Thanks,
Patrizia
--
Dr Patrizia Camelliti
Department of Physiology, Anatomy and Genetics
University of Oxford, UK
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