Hi, everybody.
I met a trouble to measure in vivo fluorescence images of S.C injected cy 5.5 tagged siRNA using IVIS imaing. The injected cy 5.5-siRnA was monitoed for its targetting to exnografted tumor whereas a strong fluorescence intensity generated by intestines under the same excitation disturbed the location of released cy5.5-siRNA.
Are there some tips to avoid this noise from intestinal organs please?
Thanks Lundvig,
I also think the autofluorescence is attributed to the diets so I will check the images agian using the mouse with fasting for at least 24h.
I would also suggest to try the alfalfa free diet first. The fluorescence lifetime of Cy5.5 is shorter than the sources of autofluorescence, but I guess that option is not possible if you are using an IVIS. I have tried spectral unmixing, the autofluorescence has a broader excitation band than the Cy5.5. However, at low concentrations of dye (similar to autofluorescence) I never really got it to work very well.
The other obvious thing is to try to place the Cy5.5 compounds in an area as far from the intestine signal as possible, and then shield the intestine. If the excitation light does not reach the area of strong autofluorescence you can avoid a lot of signal 'leakage', just as you might shield the bladder and/or liver if there is accumulation here due to excretion routes. Also try repositioning the animal and/or sheilding masks to reduce the unwanted signal. Apologies if this suggestion is too 'obvious' and you may well have already tried this.
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