DUAL CFP/YFP RATIO IMAGING

Piers Mahon psmm100 at mole.bio.cam.ac.uk
Wed May 10 07:54:32 EST 2000


I am about to try quantitative ratio imaging on a TCS using CYAN and
YELLOW fluorescent proteins with different sub-cellular location
signals.  I am expecting any positive results to be very subtle images
differences (i.e. not blue and yellow blobs in different parts of the
cell, but a cyan/yellow signal ratio that varies in sub-sections of the
same blob).

If you have any experience of CYAN FP/ YELLOW FP imaging, or related
dual imaging, please get in touch.  I'm particularly interested in
advice in three areas: 1) selecting the right filter ranges to obtain
the best bleed through / sensitivity compromise, 2) Indications of the
random noise levels I can expect in my negative controls of dual
transformants containing CYAN and YELLOW with the same location signals,
3)  Suggestions for statistical tests that I can perform to see if ratio
images with different signals on the two reporters are more variable
than images captured using the same signal.

Many thanks in advance,

Piers Mahon





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