Dear all, Carol Oxford raised a query concerning sorting chloroplasts. What you need to remember is that chloroplasts contain the natural fluorochrome chlorophyll and its 'friends'. I would therefore recommend setting your laser to 457 nm excitation (if its an argon ion) and remember to change the 90° scatter filter. As Carol pointed out, emissions can be expected at the far red end of the spectrum, giving a much larger Stokes shift than usually encountered. I have not looked at separating these two populations myself, but I would expect a dichroic filter at 45° with a cut-off in the 700-710 range would do the job. I would expect to see two populations on a plot of scatter vs fluorescence (either <710 nm or >710). Alternatively plot forward vs 90° scatter and <710 vs >710nm. In all cases use a fluorescence signal rather than a scatter signal for triggering to discriminate against debris, mitochondria etc etc. Hope this helps, look forward to hearing of your progress, reading the paper, meeting you at the Nobel prize event etc, etc. Regards Nigel Blackhall, Experimental Officer, Plant Science Division, School of Biological Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK Tel +44 115 9515151 ext 18501 Fax +44 115 9513298
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