cross talk/bleed in confocal - (Nov/01/2009 )

hello everyone,

i am new in learning confocal, i just want to ask the method on how to check and make sure that there is no or minimal cross talk or bleedthrough?

thank you so much.

There shouldn't be, the LASER light is very specific wavelengths so there should be no excitation outside of the LASER you are using. Note that some of the flurophores have similar excitation ranges, it is best to avoid using similar ones on the same sample.

Dear soymilk14,

You need to do Sequential Scanning. This means that the scans for each of the fluorophores (Dapi, FITC and TRITC) are done one by one and the software then collects them together.
Bob1 is right in that the emission spectrums for both Blue and Green, and Green and red overlap. This is a way of reducing the overlapping light. This is particularly important if you are looking at Co-localisation.

Hope this is useful.

Rhombus

rhombus on Nov 2 2009, 03:00 AM said: