DUAL CFP/YFP RATIO IMAGING

Jerry gordon at med.unc.edu
Thu May 11 08:46:52 EST 2000


Hello,
	I chose filters for emerald/topaz imaging. To choose the excitation
filters I took ratio of the relative excitation of emerald and topaz at
each wavelength. The minimum ratio occurs at the optimum wavelength for
exciting one fluorophore and the maximum ratio occurs at the optimum
wavelength for exciting the other. The same can be done for the emission
filters. This is only a starting point since the signal size will be
small with very narrow band filters. I incorporated wavelengths moving
away from the optimum. The ratio to use for multiple wavelengths is the
ratio of the sums of the relative excitations (or emissions) (the ratio
of the areas under the curves). At some point the limit on the filter
pass bands may be that the excitation and emission filter must have
enough space between them to accomodate the dichroic mirror.
	If you desire your measurements to be maximally precise you might need
to consider the possibilty of FRET between cyan and yellow FP. The
article by Gordon and Herman in Biophysical Journal May 1998 gives
methods for estimating the concentrations of two fluorophores in the
presence of FRET and crosstalk. (calculation of FRET requires estimates
of the fluorophore concentrations or the ratio of concentrations)
	HTH,
Jerry
gordon at med.unc.edu

Piers Mahon wrote:
> 
> 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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